Fusion proteins for treating cancer and related methods

ABSTRACT

Aspects of the disclosure provide fusion proteins that bind cells expressing one or more target molecules including, for example, one or more cell surface multisubunit signaling receptors (e.g., EGFRvIII-expressing cells that also express interferon receptors) and that induce anti-proliferative effects, and related compositions and methods for the treatment of cancer.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. § 371 ofinternational application PCT/US2015/028653, entitled “FUSION PROTEINSFOR TREATING CANCER AND RELATED METHODS,” filed Apr. 30, 2015 which waspublished under PCT Article 21(2) in English and which claims thebenefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No.61/986,866, entitled “CHIMERIC ACTIVATORS FOR TREATING CANCER ANDRELATED METHODS,” filed Apr. 30, 2014, the entire disclosures of each ofwhich are herein incorporated by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 28, 2016, isnamed H049870513US01-SEQLST-OMJ txt and is 54,598 bytes in size.

BACKGROUND OF INVENTION

Previous work indicated the value of combining a targeting elementhaving a high affinity for a cell surface receptor and an activityelement having a lower affinity for a second cell surface receptorthrough which signaling occurs (for example by fusion of protein domainsvia a linker). A mutation may be introduced into the activity element toreduce its receptor binding so that its binding affinity is below thatof the targeting element for its receptor. This approach has been shownto enhance the specificity of an activity element for target cellsrelative to side effect cells by as much as 20-fold, with somespecificity enhancement attributable to the attached targeting elementand the activity-reducing mutation.

However, in some therapeutic settings, the desired target cell is muchless accessible than cells through which adverse side effects aremediated. For example, solid tumors often lack lymph node drainage,therefore therapeutic proteins that enter from the circulation onlyperfuse the tumor by diffusion. This results in the concentration of thetherapeutic fusion protein being many times greater in the vicinity ofside-effect cells, such as normal tissue, compared to target cells, suchas tumor cells. In addition to tumors, targeting of proteins to thebrain also results in limited access by therapeutic proteins due to theblood-brain barrier. Therefore, there is a need in the art for improvedapproaches to targeting of therapeutic protein activities, as well asfor therapeutic proteins with enhanced specificity for their targetcells.

SUMMARY

This disclosure relates to protein engineering and construction offusion proteins. Some aspects of the present disclosure focus onimproving the properties of a class of engineered fusion proteins termed“chimeric activators.” These proteins include a targeting element thatbinds to a cell surface receptor, an activity element that binds to adistinct receptor on the same cell, and a linker connecting the twoprotein domains. In some embodiments, the activity element has reducedactivity due to the presence of one or more amino acid substitutions. Insome embodiments, decreasing the binding affinity of the activityelement in an effort to reduce undesired side effects (e.g.,anti-proliferative or cytotoxic effects on non-target cells) can renderthe chimeric molecules ineffective due to weak receptor binding, suchthat the targeting receptor-bound chimeric activator may be internalizedand degraded through normal membrane clearance phenomena beforesignaling has a chance to occur. Aspects of the present disclosure arebased in part on the recognition that many signaling molecules bind tomore than one receptor or receptor subunit, the interactions with thesereceptors vary in strength by several orders of magnitude, and thatthese differences can be taken into account when designing or producinga chimeric activator as described herein.

In some embodiments, aspects of the disclosure relate to fusion proteinscomprising a first protein domain that binds to a multisubunit signalingreceptor (e.g., a cell surface multisubunit signaling receptor), asecond protein domain (e.g., an antibody variable region element, aligand, or other peptide) that binds to a cell surface antigen, and alinker that connects the first protein domain and the second proteindomain, wherein the first protein domain that binds to the multimericsignaling receptor includes a first amino acid substitution and a secondamino acid substitution such that the binding affinity of the firstprotein domain to a first subunit of the multisubunit signaling receptoris altered by the first amino acid substitution and the binding affinityof the first protein domain to a second subunit of the multisubunitsignaling receptor is altered by the second amino acid substitution.

In some embodiments, the fusion protein comprises one polypeptide chain.In some embodiments, the multisubunit signaling receptor and the cellsurface antigen are expressed on at least one cell type in a human. Insome embodiments, the first mutation decreases the binding affinity ofthe first protein domain for the first receptor subunit and the secondmutation increases the affinity of the first protein domain for thesecond receptor subunit. In some embodiments, the multisubunit signalingreceptor is a Type 1 interferon receptor. In some embodiments, theprotein domain that binds to a multisubunit signaling receptor is acytokine or hormone. In some embodiments, this protein domain comprisesa Type 1 interferon. In some embodiments, the Type 1 interferoncomprises one or more amino acid substitutions selected from the groupconsisting of L30A, R145A, M149A, E59A, H58A, and R150A.

In some embodiments, an antibody variable region element comprises anantibody variable region or regions or a derivative or fragment thereofthat is capable of binding a peptide provided by the amino acid sequenceLys-Gly-Asn-Tyr-Val-Val-Thr-Asp-His (SEQ ID NO: 17). In someembodiments, the protein domain that binds to the multi-subunitsignaling receptor comprises the amino acid sequence provided by SEQ IDNO: 18.

In some embodiments, the linker connects the C-terminal end of secondprotein domain (e.g., the antibody or antibody variable region element)to the N-terminal end of the first protein domain. In some embodiments,the linker connects the C-terminal end of the first protein domain tothe N-terminal end of the second protein domain (e.g., the antibody orantibody variable region element). In some embodiments, the firstprotein domain inhibits cellular proliferation. In some embodiments, theantibody variable region element comprises a heavy chain variable regioncomprising the amino acid sequence provided by SEQ ID NO: 15 and a lightchain variable region comprising the amino acid sequence provided by SEQID NO: 16. In some embodiments, the Type 1 interferon comprisesmutations H58A and R150A. In some embodiments, the Type 1 interferoncomprises mutations E59A and M149A.

In some embodiments, aspects of the disclosure relate to a fusionprotein (e.g., a chimeric activator) comprising a polypeptide that bindsto a heteromultimeric receptor comprising at least a first subunit and asecond subunit, a second protein domain (e.g., an antibody variableregion element, a ligand, or other peptide) that binds to a target cellsurface receptor (e.g., EGFRvIII), and a linker that connects thepolypeptide and the antibody variable region element. In someembodiments, the polypeptide that binds to the heteromultimeric receptorincludes one or more amino acid substitutions such that the bindingaffinity of the polypeptide to a first subunit of the heteromultimericreceptor is increased and/or the binding affinity of the polypeptide toa second subunit of the heteromultimeric receptor is decreased (e.g.,such that the relative binding affinities of the polypeptide to eachreceptor is less than 10 fold, 5 fold, 2 fold, or 1.5 fold, or 1.1 foldof each other). In some embodiments, a fusion protein includes apolypeptide (e.g., a first protein domain) having at least one aminoacid substitution such that the binding affinity of the polypeptide toat least one subunit of the heteromultimeric receptor is increasedrelative to binding affinity of the unsubstituted polypeptide.

In the case of Type 1 interferons, the interaction with the Type 1interferon receptor subunit 2 (IFNAR2) is much stronger than with theType 1 interferon receptor subunit 1 (IFNAR1). In a binding modelcontemplated by the present disclosure, a polypeptide such as aninterferon (IFN) initially bind to IFNAR2, and then the IFN/IFNAR2complex diffuses in two dimensions in the cell membrane until it findsIFNAR1. A stable signaling complex is formed after formation of theIFNAR1/IFNAR2/IFN trimeric complex, and subsequent intracellular eventsoccur such as binding of downstream signaling proteins andphosphorylation. According to this model, the IFNAR1/IFNAR2/IFN trimericcomplex may dissociate before intracellular events occur.

In some embodiments, the polypeptide that binds to the Type 1 interferonreceptor comprises a Type 1 interferon-α region having one or moresubstitution mutations in a portion of the polypeptide that interactswith the receptor. For example, in some embodiments one or moresubstitution mutations are selected from the group consisting of L30A,R145A, M149A, E59A, H58A, and R150A (e.g., both M149A and E59A and/orboth H58A and R150A).

In some embodiments, the heteromultimeric receptor is a Type 1interferon receptor. In some embodiments, the amino acid sequence of thepolypeptide that binds to the heteromultimeric receptor is an interferonand comprises one or more amino acid substitutions that improveexpression, stability (e.g., structural stability and/or resistance tointracellular degradation for example associated with cellularinternalization), or signaling of the polypeptide. In some embodiments,such substitutions in the polypeptide backbone result in the presence ofone or more of Arg at residue 23, Pro at residue 26, Asp at residue 44,Gln at residue 52, Ala at residue 53, Ser at residue 55, Glu at residue83, Thr at residue 101, Val at residue 104, Gly at residue 105, Glu atresidue 107, and/or Glu at residue 125 as described herein. In someembodiments, one or more of these backbone substitutions may enhancebinding of the polypeptide to the first and second subunit of theheteromultimeric receptor and increase stability of the trimericsignaling complex. In some embodiments, one or more of thesesubstitutions increase resistance of the trimeric complex tointernalization and degradation.

In some embodiments, the antibody variable region element that binds toEGFRvIII comprises MR1-1 or a derivative or fragment thereof. In someembodiments, the linker has a net charge.

In some embodiments, aspects of the disclosure relate to fusion proteinscomprising a protein domain that binds to a multisubunit signalingreceptor, a second protein domain (e.g., an antibody variable regionelement) that binds to a cell surface antigen, and a linker thatconnects the protein domain and the second protein domain (e.g., theantibody variable region element), wherein the linker is a peptidelinker and has a net charge. In some embodiments, the net charge of thelinker is negative. In some embodiments, the linker comprises aminoacids selected from the group consisting of glycine, serine, glutamate,and aspartate. In some embodiments, the net charge of the linker ispositive. In some embodiments, the linker comprises amino acids selectedfrom the group consisting of lysine, arginine, and histidine. In someembodiments, the linker comprises 10 to 200 amino acids in length. Insome embodiments, the linker comprises a repeat of GGGSE (SEQ ID NO:11),GSESG (SEQ ID NO:12), or GSEGS (SEQ ID NO:13). In some embodiments, thelinker comprises the sequence of GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS(SEQ ID NO:14).

In some embodiments, the linker comprises an amino acid sequencecomprising regularly spaced negatively charged amino acids andnon-regularly spaced non-charged amino acids. In some embodiments, thenon-charged amino acids are selected from the group consisting ofglycine, serine, alanine, proline, and threonine. In some embodiments,the non-charged amino acids are selected from the group consisting ofglycine and serine.

In some embodiments, the protein domain that binds to the multisubunitsignaling receptor is a cytokine or hormone. In some embodiments, theprotein domain that binds to the multi-subunit signaling receptor is aType 1 interferon. In some embodiments, the protein domain that binds tothe multisubunit signaling receptor comprises the amino acid sequenceprovided by SEQ ID NO: 18. In some embodiments, the linker connects theC-terminal end of the antibody variable region element to the N-terminalend of the protein domain. In some embodiments, the linker connects theC-terminal end of the protein domain to the N-terminal end of theantibody or antibody variable region element. In some embodiments, theprotein domain inhibits cellular proliferation.

In some embodiments, the antibody variable region element comprises aheavy chain variable region comprising the amino acid sequence providedby SEQ ID NO: 15 and a light chain variable region comprising the aminoacid sequence provided by SEQ ID NO: 16. In some embodiments, the Type 1interferon comprises mutations H58A and R150A. In some embodiments, theType 1 interferon comprises mutations E59A and M149A.

In some embodiments, the fusion protein selectively binds to cancercells relative to non-cancer cells. In some embodiments, the fusionprotein selectively binds to cancer cells that express EGFRvIII relativeto cells that do not express EGFRvIII. Accordingly, some aspects of thedisclosure relate to a chimeric activator comprising a polypeptide thatbinds to a Type 1 interferon receptor, an antibody that binds toEGFRvIII, and a linker that connects the polypeptide and the antibody.In some embodiments, the linker is a peptide linker and has a netcharge. In some embodiments, the polypeptide that binds to the Type 1interferon receptor comprises one or more amino acid substitutions thatimprove expression, stability, or signaling of the polypeptide. In someembodiments, the polypeptide that binds to the Type 1 interferonreceptor comprises a Type 1 interferon-α (IFN-α) region having one ormore substitution mutations selected from the group consisting of L30A,R145A, M149A, E59A, H58A, and R150A (e.g., both M149A and E59A and/orboth H58A and R150A). In some embodiments, the antibody that binds toEGFRvIII comprises MR1-1 or a derivative or fragment thereof.

In some embodiments, the Type 1 interferon-α comprises one or more aminoacid substitutions in the polypeptide that result in Arg at residue 23,Pro at residue 26, Asp at residue 44, Gln at residue 52, Ala at residue53, Ser at residue 55, Glu at residue 83, Thr at residue 101, Val atresidue 104, Gly at residue 105, Glu at residue 107, and/or Glu atresidue 125, or any combination thereof.

In some embodiments, the linker connects the C-terminal end of theantibody or antibody variable region element to the N-terminal end ofthe polypeptide. In other embodiments, the linker connects theC-terminal end of the polypeptide to the N-terminal end of the antibodyor antibody variable region element.

In some embodiments, the antibody or antibody variable region elementthat binds to EGFRvIII comprises a svFc, sdAb, Fab, Fab2, or a fulllength immunoglobulin (e.g., a full length immunoglobulin chain, forexample a heavy chain and/or a light chain). In some embodiments, theantibody or antibody variable region element comprises a heavy chainvariable region of SEQ ID NO: 15 and a light chain variable region ofSEQ ID NO: 16.

In some embodiments, the Type 1 interferon comprises mutations H58A andR150A In other embodiments, the Type 1 interferon comprises mutationsE59A and M149A.

In some embodiments, the linker comprises 10 to 200 amino acids inlength. In some embodiments, the net charge of the linker is negative.In some embodiments, the linker comprises amino acids selected from thegroup consisting of glycine, serine, glutamate, and aspartate. In someembodiments, the linker comprises a repeat of GGGSE (SEQ ID NO:11),GSESG (SEQ ID NO:12), or GSEGS (SEQ ID NO:13). In some embodiments, thelinker comprises the sequence GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS (SEQID NO:14).

In other embodiments, the net charge of the linker is positive. In otherembodiments, the linker comprises amino acids selected from the groupconsisting of lysine, arginine, and histidine.

In some embodiments, the chimeric activator selectively binds to cancercells relative to non-cancer cells. In some embodiments, the chimericactivator protein selectively binds to cancer cells that expressEGFRvIII relative to cells that do not express EGFRvIII.

Aspects of the disclosure relate to isolated proteins comprising SEQ IDNO: 18.

Accordingly, in some embodiments aspects of the disclosure relate to anisolated chimeric activator protein comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 1 [MR1-1 INFa 2-1b WT],SEQ ID NO: 2 [MR1-1 INFa 2-1B L30A], SEQ ID NO: 3 [MR1-1 INFa 2-1BR145A], SEQ ID NO: 4 [MR1-1 INFa 2-1b E59A M149A], and SEQ ID NO:5[MR1-1 INFa 2-1b H58A R150A].

Aspects of the disclosure relate to isolated nucleic acids that encodeany of the fusion proteins described above and elsewhere here. In someembodiments, the isolated nucleic acid comprises in frame a firstsequence encoding a protein domain that binds to a multisubunitsignaling receptor, a second sequence encoding an antibody variableregion element that binds a cell surface antigen, and a third sequenceencoding a linker that connects the protein domain and the antibodyvariable region element, wherein the linker is a peptide linker and hasa net charge.

Other aspects of the disclosure relate to an isolated nucleic acid thatencodes a chimeric activator protein as described above or elsewhereherein.

In some embodiments, aspects of the disclosure relate to an isolatednucleic acid encoding a chimeric activator comprising in frame a firstsequence encoding a polypeptide that binds to a Type 1 interferonreceptor, a second sequence encoding an antibody that binds to EGFRvIII,and a third sequence encoding a linker that connects the polypeptide andthe antibody. In some embodiments, the linker is a peptide linker andhas a net charge. In some embodiments, the polypeptide that binds to theType 1 interferon receptor comprises a Type 1 interferon-α (IFN-α)region having one or more substitution mutation selected from the groupconsisting of L30A, R145A, M149A, E59A (e.g., both M149A and E59A),H58A, and R150A (e.g., both H58A and R150A). In some embodiments, theantibody that binds EGFRvIII comprises MR1-1 or a derivative or fragmentthereof.

Aspects of the disclosure relate to methods for manufacturing a fusionprotein comprising a protein domain that binds to a multisubunitsignaling receptor, an antibody variable region element that binds to acell surface antigen, and a linker that connects the protein domain andthe antibody variable region element, comprising mutating the proteindomain such that the binding affinity of the protein domain to a firstsubunit of the multisubunit signaling receptor is increased and thebinding affinity of the protein domain to a second subunit of themultisubunit signaling receptor is decreased relative to an unmutatedprotein domain; selecting a linker length and an amino acid compositionthat enhances the specificity of the fusion protein for cells bearingthe cell surface antigen; and preparing the fusion protein. In someembodiments, the first subunit is IFNAR1 and the second subunit isIFNAR2.

Other aspects of the disclosure relate to methods for manufacturing achimeric activator comprising a polypeptide that binds to the Type 1interferon receptor, an antibody fragment that binds to EGFRvIII, and alinker that connects the polypeptide and the antibody fragment. In someembodiments, the method involves mutating the polypeptide such that thebinding affinity of the polypeptide to a first subunit of the Type 1interferon receptor is increased and the binding affinity of thepolypeptide to a second subunit of the Type 1 interferon receptor isdecreased. In some embodiments, the method involves selecting a linkerlength and/or an amino acid composition that stabilizes and/or increasesthe activity of the chimeric activator (e.g., after internalization). Insome embodiments, the method involves preparing a chimeric activator. Insome embodiments, the chimeric activator binds to two subunits of a Type1 interferon receptor. In some embodiments, the first subunit is IFNAR1and the second subunit is IFNAR2.

Aspects of the disclosure relate to methods for targeted inhibition ofcellular proliferation comprising contacting a cell with an effectiveamount of any of the fusion proteins described above and elsewhereherein. In some embodiments, the cell is characterized by expression ofEGFRvIII. In some embodiments, the cell is in a human. In someembodiments, the cell is obtained from a human.

Some aspects of the disclosure relate to methods for targeted inhibitionof cellular proliferation. In some embodiments, the method involvescontacting a cell with an effective amount of a fusion protein (e.g., achimeric activator). In some embodiments, the method involves assessingcellular proliferation after contacting the cell with the fusion protein(e.g., chimeric activator). In some embodiments, the cell ischaracterized by expression of EGFRvIII. In some embodiments, the cellis in an individual (e.g., in situ, in vivo). In other embodiments, thecell is obtained from an individual (e.g., ex vivo).

Aspects of the disclosure relate to methods for treating cancercomprising administering to an individual having cancer an effectiveamount of any of the fusion proteins described above or elsewhereherein. In some embodiments, the cancer is characterized by EGFRvIIIexpression. In some embodiments, the cancer is glioblastoma. In someembodiments, the individual has received at least one cancer treatmentselected from the group consisting of surgery, chemotherapy, andradiation therapy. In some embodiments, the individual is concurrentlyadministered the fusion protein and at least one cancer treatmentselected from the group consisting of surgery, chemotherapy, andradiation therapy.

Some aspects of the disclosure relate to methods for treating cancer. Insome embodiments, the method involves administering to an individualhaving cancer an effective amount of a fusion protein (e.g., a chimericactivator). In some embodiments, the cancer is characterized by EGFRvIIIexpression. In some embodiments, the cancer is glioblastoma.

Other aspects of the disclosure relate to methods for treating anindividual having cancer. In some embodiments, the method involvesdetecting whether the cancer expresses EGFRvIII, and if EGFRvIIIexpression is detected, administering to the individual an effectiveamount of a chimeric activator.

Yet other aspects of the disclosure relate to compositions comprisingany of the fusion proteins described above or elsewhere herein. In someembodiments, the composition also comprises a pharmaceuticallyacceptable carrier, wherein the pharmaceutically acceptable carriercomprises a phosphate-buffered saline or a buffer comprising a sugar,arginine, citrate, and/or a Tween compound.

Other aspects of the disclosure provide compositions comprising achimeric activator as described herein. In some embodiments, thecomposition further comprises a pharmaceutically acceptable carrier. Insome embodiments, the pharmaceutically acceptable carrier comprises aphosphate-buffered saline or a buffer comprising a sugar, arginine,citrate, and/or a Tween compound.

BRIEF DESCRIPTION OF DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure, which can be better understood by reference to one of moreof these drawings in combination with the detailed description of thespecific embodiments presented herein.

FIG. 1 presents a set of example fusion proteins comprising a targetingelement (T) and an activity element (A) connected by a negativelycharged linker (indicated by a line). FIG. 1A presents the generalstructure of a fusion protein comprising an activity element, anegatively charged linker, and a targeting element. FIG. 1B presents anexample of a fusion protein comprising an activity element, a negativelycharged linker, and an activity element consisting of a single-chain Fv(scFv) that includes a linker between the VH and VL domains. FIG. 1Cpresents an example of a fusion protein comprising an activity elementwith a mutation (indicated by an “X”) that reduces its binding to areceptor, a negatively charged linker, and an activity elementconsisting of an (scFv) that includes a linker between the VH and VLdomains. FIG. 1D presents an example of a fusion protein comprising anactivity element fused to a “SNAP” protein domain, a negatively chargedlinker consisting of a nucleic acid attached at one end to the SNAPdomain, and an activity element consisting of a second SNAP domain fusedto a single-chain Fv (scFv) with a linker between the VH and VL domainssuch that the second SNAP domain is also attached to the other end ofthe nucleic acid. FIG. 1E is a schematic demonstrating the repulsiveelectrostatic interaction between the surface of a cell, which isnegatively charged, and negative charges on a linker in a fusion proteinthat includes an activity element and a targeting element.

FIG. 2 presents example sequences of a negatively charged peptidelinker, consisting of glycine, serine, and glutamic acid residues. FIG.2A shows a fusion protein with the negatively charged linker highlightedby a bracket. FIG. 2B presents an exemplary 35-amino acid linkersequence (SEQ ID NO. 14) with randomly placed glycine and serineresidues and glutamic acid residues at regular five amino acidintervals. The DNA sequence corresponds to SEQ ID NO: 26. FIG. 2Cpresents an exemplary 35-amino acid linker sequence with randomly placedglycine and serine residues and glutamic acid residues at regularfour-amino acid intervals (SEQ ID NO: 27). The DNA sequence correspondsto SEQ ID NO: 28. The coding sequences of the linkers are also shown andindicate how non-repetitive encoding is accomplished.

FIG. 3 presents example fusion proteins with an activity element (A), alinker (indicated by a line), and a targeting element (T), in which theactivity element binds to at least two different receptor subunits on acell surface to achieve signaling; and the binding is through twodifferent faces whose receptor subunit binding can be independentlymodulated. FIG. 3A presents an example fusion protein that issimultaneously bound to a targeting receptor (Rec_(T)) and an activityelement receptor with two subunits (R1_(A) and R2_(A)). FIG. 3B presentsthe general structure of a fusion protein with an activity element, alinker, and a targeting element, in which the activity element containsone or more mutations (indicated by an “X”) on each protein face thatinteracts with one of the receptor subunits (and thus contains at leasttwo mutations). FIG. 3C presents an example fusion protein in which themutation(s) on one face of the activity element have the net effect ofincreasing binding to a receptor subunit (indicated by an “X+”), andmutations on another face also have the effect of increasing binding butto a distinct receptor subunit (also indicated by an “X+”). FIG. 3Dpresents an example fusion protein in which the mutation(s) on one faceof the activity element have the net effect of increasing binding to areceptor subunit (indicated by an “X+”), and mutations on another facehave the effect of decreasing binding to a distinct receptor subunit(indicated by an “X−”). FIG. 3E presents such an example fusion proteinin which the mutation(s) on one face of the activity element have thenet effect of decreasing binding to a receptor subunit (indicated by an“X”), and mutations on another face also have the effect of decreasingbinding but to a distinct receptor subunit (indicated by an “X”). FIG.3F shows a table of the binding dissociation constants for wild-type andmutant forms of IFN-α used in fusion proteins described in the Examplesand FIG. 5, with respect to IFNAR1 (“R1”) and IFNAR2 (“R2”). In fusionproteins that contain double mutations (e.g., His57Ala (H57A)Arg149Ala(R149A) and Glu58Ala (E58A) Met148Ala (M148A)), one mutation decreasesbinding while the other increases binding, such that the bindingaffinity of each face of IFNα with its receptor subunit is substantiallythe same.

FIG. 4 shows an alignment of human INFα sequences, with the sequence ofIFNα2-1b at the top in bold. The sequences, from top to bottom,correspond to SEQ ID NOs: 18, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, and 29.

FIG. 5 presents exemplary viability curves of wild-type U87 cells(diamonds) or U87 cells that express EGFRvIII (U87 EGFRvIII; squares)following treatment for 72 hours with the indicated fusion proteinsproduced in Pichia pastoris supernatants. FIG. 5A shows viabilityfollowing treatment with wild-type INFα. FIG. 5B shows viabilityfollowing treatment with MR1-1 IFNα 2-1b wt. FIG. 5C shows viabilityfollowing treatment with MR1-1 IFNα 2-1b L30A. FIG. 5D shows viabilityfollowing treatment with MR1-1 IFNα 2-1b R145A. FIG. 5E shows viabilityfollowing treatment with MR1-1 INFα 2-1b H58A R150A. FIG. 5F showsviability following treatment with MR1-1 IFNα 2-1b E59A M149A.

FIG. 6 shows an SDS-PAGE gel quantifying MR1-1-IFNα2-1b(R145A)production. Lane 1 contains a size standard. In lanes 2-5, 5, 2.5, 1.25and 0.75 micrograms of bovine serum albumin (BSA) was run; the BSA isindicated by “1” and the white box. Bands in lanes 6 and 7 are samplesof MR1-1-IFNα2-1b(R145A) diluted by 5-fold and 10-fold, respectively,following purified by cobalt affinity via the His6 tag and then by fastprotein liquid chromatography (FPLC). Protein bands of the predictedsize are indicated by “2” and “3”. Lane 8 is the undiluted supernatantfrom the initial Pichia pastoris culture; and “4” represents the proteinband at the predicted size. “5” indicates contaminating protein bandsthat constitute less than 10% of the total protein in the sample. Theresults indicate that the MR1-1-IFNα2-1b (R145A) protein can be purifiedfrom Pichia pastoris cultures using conventional methods.

FIG. 7 shows an FPLC profile of MR1-1 INFα2-1b(R145A). The arrowsindicate the main peak and a shoulder peak representing MR1-1 IFNα2-1b(R145A). When fractions corresponding to the peak and the shoulder peakwere examined by SDS-PAGE gel, both showed an identical patternconsisting of >90% MR1-1-INFα2-1b (R145A) similar to that of FIG. 6.This result suggests that because the loaded sample was in a highlyconcentrated state, the scFv portion of MR1-1 INFα2-1b (R145A) was in amonomer-dimer equilibrium and that the shoulder represents non-covalentdimeric MR1-1 IFNα2-1b (R145A).

FIG. 8 shows results of a growth inhibition assay in which murineNeuro-2a cells (N2a cells) were treated with between approximately 1picomolar to 1 micromolar murine IFN-α2 (muIFN WT), human IFN-α2a (huIFNWT), or MR1-1 INFα2-1b. The results indicate that the murine protein wasmost active in growth inhibition of the murine N2a cells, MR1-1 IFNα2-1bwas detectably active but less so than the murine INFα, and that thehuman IFNα2a protein was not detectably active.

FIG. 9 shows tumor volume in mice at the indicated time points. Thearrows indicate administration of the fusion protein (MR1-1-INFα2-1b(R145a) at 135 mcg/mouse/day or vehicle control (PBS). Tumors volumeswere calculated by the formula (length×width²)/2. Squares are micetreated with the fusion protein; and circles mice that received thecontrol.

DETAILED DESCRIPTION OF INVENTION

Aspects of the disclosure are based on the recognition that coupling amolecule with a desired activity to a targeting molecule can be aneffective method for targeting the activity to cells expressing atargeted feature. However, in some therapeutic settings, the targetedcell is less accessible than cells not expressing the targeted feature.Such cells are referred to herein as “side effect cells.” The desiredactivity performed on a side effect or non-targeted cell mediates manyof the side effects of such therapeutic molecules. For example, solidtumors often lack lymph node drainage and therapeutic molecules onlyperfuse the tumor by diffusion. This results in a local concentration ofthe therapeutic molecule that is many times greater in the vicinity ofside effect cells compared to target cells, such as tumor cells. Inanother example, therapeutic molecules that target molecules expressedby cells of the brain must cross the blood-brain barrier. A relativelylow proportion of the therapeutic molecule succeeds in accessing thislimited site, resulting in a much lower concentration of the therapeuticmolecule contacting the targeted cells compared to the side effectcells. Accordingly, there is a need for improved and novel approaches toenhance the specificity and the desired activity of therapeuticmolecules to targeted cells while minimizing any activity towards sideeffect cells.

Described herein are novel fusion proteins that are capable of bindingtwo distinct targets on a cell, wherein one of the targets is amultimeric target or multi-subunit signaling protein (e.g., aheteromultimeric target). In some embodiments, the fusion proteins arealso targeted to cells expressing a cell surface antigen, such as anEGFR variant, e.g., EGFRvIII, that is associated with diseases mediatedby constitutive EGFR signaling, such as cancer. For example, upontargeting to EGFRvIII-expressing cells, fusion proteins can also bind toa multisubunit signaling receptor, such as a heteromultimeric receptor,e.g., a Type 1 interferon receptor (IFNAR). Other examples ofheteromultimeric receptors that can be bound by fusion proteins include,without limitation, the IL-2 receptor, IL-4 receptor, and LIF receptor.Binding of fusion protein to a multisubunit signaling receptor caninduce a desired activity in the targeted cell. For example, some fusionproteins described herein induce an anti-proliferative effect on thetargeted cell and reduce undesired activity on side effect cells,providing an improved therapeutic approach to cancer treatment. In someembodiments, described herein are fusion proteins comprising an antibodyregion that binds EGFRvIII, a protein domain that binds to the Type 1interferon receptor, and a linker that connects the antibody region andthe protein domain, and uses thereof for inducing anti-proliferativeeffects in cells expressing EGFRvIII and treating disease associatedwith constitutive EGFR signaling due to expression of EGFRvIII. Thefusion proteins described herein exhibit enhanced specificity andtherapeutic activity in target cells and reduce undesired targeting ofside effect cells (e.g., normal, non-cancer cells).

In some embodiments, the fusion proteins described herein are chimericactivators As used herein, a “chimeric activator” refers to anengineered protein that binds to one or more receptors or antigens onthe surface of a cell. A chimeric activator includes an “activityelement,” a “targeting element,” and a polypeptide linker that connectsthe activity element and the targeting element. The activity element ofthe chimeric activator binds to a receptor on a target cell surface andhas a biological activity, such as initiation of a signal transductionpathway that may result in a desired effect. The targeting element bindsto a targeting receptor on the surface of the same cell, such as a cellsurface antigen. The polypeptide linker connects the activity elementand the targeting element such that both the activity and targetingelements can simultaneously bind to their receptors/antigens on thesurface of the same cell. In some embodiments, the chimeric activatorsdescribed herein also comprise at least one mutation in the activityelement that reduces its biological activity relative to the naturalprotein or protein domain from which it was derived.

As used herein, a “protein domain” or “domain” refers to a distinctglobular unit that can be identified as such by a structuredetermination method such as X-ray crystallography or NMR, by otherbiophysical methods such as scanning calorimetry according to which aprotein domain melts as a distinct unit (see for example Pabo et al.Proc Natl Acad Sci USA. (1979) 76(4):1608-12), or by sequence similarityto protein domains whose structure has been determined. The SCOPdatabase (Murzin et al. J Mol Biol. (1995) 247(4):536-40) provides theidentification of protein domains so that the domain organization of anew protein can be identified by sequence comparison. Protein domainscomprise an amino acid sequence that is sufficient to drive folding ofsuch a polypeptide into a discrete structure, in which essentially allof the rotatable bonds along the main chain of the polypeptide areconstrained to within about 10 degrees. In contrast, linkers, shortpeptides, molten globules, and unstructured segments are examples ofpolypeptides that are not domains and do not have these characteristics.

Fusion Proteins

The present disclosure provides novel fusion proteins for treatingcancer. As used herein, the term “fusion protein” refers to any proteinor polypeptide that is comprised of peptides, polypeptides, or proteindomains from at least two different sources (e.g. two differentproteins). The term fusion protein also encompasses “chimericactivators.” The fusion proteins are capable of simultaneously bindingto multiple unrelated receptors on the same target cell and induce adesired activity. As described herein, the fusion proteins comprise afirst protein domain that binds to a multi-subunit signaling receptor,referred to as an activity element; a second protein domain (e.g., anantibody variable region element, a ligand, or other peptide) that bindsto a cell surface receptor, referred to as a targeting element; and alinker that connects the activity element and the targeting element. Insome embodiments, the activity element is mutated or altered so it hasmodified (e.g., increased or decreased) activity relative to itsnaturally occurring counterpart. One portion of the fusion proteinfunctions as a targeting element and another portion of the fusionprotein functions as the activity element. In some embodiments, thefusion protein is a chimeric activator comprising a protein domain thatbinds to Type 1 interferon receptor (IFNAR), an antibody that binds toEGFRvIII, and a linker that connects the protein domain and theantibody.

Type 1 interferons (IFN) are a subset of interferon proteins that bindto the Type 1 interferon receptor (IFNAR) complex. The IFNAR consists oftwo subunits IFNAR1 and IFNAR2, that upon ligand binding activate theJanus kinase (Jak) and signal transduction and activator oftranscription (STAT) signaling pathway to induce expression of more than400 IFN-regulated genes. In mammals, IFNs are further subdivided intogroups IFN-α, IFN-β, IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω, and IFN-ζ. IFN-αis produced by leukocytes and has at least 13 subtypes including IFN-α1,IFN-α2, IFN-α4, IFN-α5, IFN-α6, IFN-α7, IFN-α8, IFN-α10, IFN-α13,IFN-α14, IFN-α16, IFN-α17, and IFN-α21. IFN-β is predominantly producedby fibroblasts has two subtypes: IFN-β1 and IFN-β3. Both IFN-α and IFN-βare involved in the innate immune response, and upon binding to theIFNAR complex induce anti-proliferative effects and the canonicalanti-viral response. Accordingly, in some embodiments, a chimericactivator can comprise any one of Type 1 interferons such that it bindsto the IFNAR1 subunit and IFNAR2 subunit.

Epidermal growth factor receptor (EGFR) is cell surface receptor thatbelongs to the ErbB family of receptors. EGFR binds ligands includingepidermal growth factor (EGF) and transforming growth factor-α (TGF-α).Upon ligand binding, EGFR dimerizes and autophosphorylates tyrosineresidues in the intracellular C-terminal portion of the receptor, whichtriggers a signaling cascade that results in expression of genesinvolved in modulating cell migration, adhesion, and proliferation. Asused herein, “EGFRvIII” refers to EGFR variant type III that is produceddue to an in-frame deletion of exons 2-7, which also generates a novelglycine residue at the new junction between exons 1 and 8. The deletioncorresponds to removal of 267 amino acids of the extracellular domain ofEGFR and an inability of EGFRvIII to bind its ligands. Despite the lossof ligand binding capability, EGFRvIII has been found to bephosphorylated and constitutively active, leading to sustainedactivation of anti-apoptotic and pro-invasive signaling pathways.Further enhancing the oncogenic and tumorigenic potential of cellsexpressing the EGFR variant, EGFRvIII has impaired internalization anddegradation. In some embodiments, chimeric molecules described hereinspecifically bind to EGFRvIII. In some embodiments, chimeric moleculesdescribed herein include an antibody region that specifically binds toEGFRvIII. However, it should be appreciated that in some embodiments achimeric molecule described herein can include a peptide (e.g., aligand) that specifically binds to EGFRvIII instead of an antibodyregion.

As used herein, a “target cell” refers to a cell that expresses a targetprotein of interest (e.g., EGFRvIII). In some embodiments, the cell hasone or more additional characteristics of a tumorigenic or cancer cell.As also used herein, a “targeting molecule” refers to a molecule on thecell surface that identifies the cell as a target for a desiredactivity. The targeting molecule can be detected (e.g., bound) by aportion of the chimeric activator. In some embodiments, expression andcell surface localization of EGFRvIII is the targeting molecule that isbound by the chimeric activator.

Protein Domains that Bind a Multisubunit Signaling Receptor

In some aspects of the disclosure, the fusion protein comprises aprotein domain that binds to one or more subunits of a multisubunitsignaling receptor. The protein domain that binds to one or moresubunits of a multisubunit signaling receptor may also be referred to asan activity element. As used herein, the terms “multisubunit signalingreceptor,” “multimeric receptor,” and “multimeric target” may be usedinterchangeably and refer to a receptor on the surface of a cell that iscomprised of two or more subunits.

In some embodiments, the multisubunit signaling receptor is ahomomultimeric receptor. In other embodiments, the multisubunitsignaling receptor is a heteromultimeric receptor. In some embodiments,the protein domain of the fusion protein binds to at least two subunitsof the multimeric receptor. In some embodiments, the multisubunitreceptor is a metazoan signaling receptor.

As used herein, a “face” of a protein or protein domain refers to asurface of a protein. A protein or protein domain that binds to anothermolecule, such as a multisubunit receptor, by different faces of theprotein domain means different amino acid residues of the protein domaininteract with the other molecule. In some embodiments, the proteindomain of the fusion protein binds to the multisubunit receptor bydifferent faces of the protein domain. In some embodiments, one face ofthe protein domain of the fusion protein binds to one subunit of themultisubunit receptor and another face of the protein domain binds toanother subunit of the multisubunit receptor.

In some embodiments, the protein domain that binds to the multisubunitsignaling receptor is a cytokine or a hormone. In some embodiments, themultisubunit signaling receptor is the Type 1 interferon receptor. Insome embodiments, the fusion protein comprises a protein domain thatbinds to the Type 1 interferon receptor (IFNAR). Preferably, the proteindomain that binds to IFNAR activates the IFN signaling pathway andinduces anti-proliferative effects. In some embodiments, the polypeptidebinds to IFNAR and induces death of the target cell (e.g., cytotoxiceffects). The anti-proliferative effects of the fusion protein or thechimeric activator can be evaluated by methods described herein orroutine in the art.

As used herein, “IFNAR” or “IFNAR complex” refers to the combination ofthe IFNAR1 and IFNAR2 subunits that together comprise a functional Type1 interferon receptor. In some embodiments, a protein domain that bindsto the IFNAR complex binds first to the IFNAR1 subunit and then theIFNAR2 subunit. In some embodiments, the protein domain that binds tothe IFNAR complex binds first the IFNAR2 subunit and then to the IFNAR1subunit. It is known in the art that a polypeptide will bind first to areceptor or receptor subunit to which the polypeptide has the highestbinding affinity. In some embodiments, the protein domain has a higherbinding affinity to IFNAR2 compared to IFNAR1. Without wishing to bebound by any particular theory, the polypeptide portion of chimericactivator binds first to IFNAR2. The polypeptide/IFNAR2 complex candiffuse in two dimensions in the cell membrane until it contacts aIFNAR1 subunit. The IFNAR1/IFNAR2/polypeptide trimeric complex is asignaling complex.

Other examples of multimeric signaling receptors include, withoutlimitation. IL-2 receptor, IL-4 receptor, and LIF receptor. In someembodiments, the protein domain that binds to the multimeric signalingreceptor is a cytokine or a hormone.

Any polypeptide or protein domain that binds to IFNAR can be used in thechimeric activators described herein. In preferred embodiments, theprotein domain that binds IFNAR is a Type 1 interferon (IFN). In someembodiments, the IFN is an IFN-α selected from IFN-α1, IFN-α2, IFN-α4,IFN-α5, IFN-α6, IFN-α7, IFN-α8, IFN-α10, IFN-α13, IFN-α14, IFN-α16,IFN-α17, or IFN-α21. In some embodiments, the IFN is IFN-α2. In someembodiments, the IFN is an IFN-β selected from IFN-β1 or IFN-β3.

In some embodiments, the protein domain that binds to the IFNAR complexis a synthetic IFN. The amino acid sequence of a synthetic IFN may begenerated by comparing the amino acid sequence of multiple (e.g., two ormore) IFN and identifying a preferred amino acid sequence. In someembodiments, the amino acid sequence of a synthetic IFN may be generatedby including one or more amino acid substitutions that stabilize and/orincrease the intracellular activity of the chimeric activator. In someembodiments, the protein domain that binds to the IFNAR complex isIFNα2-1 and is provided by SEQ ID NO: 18.

As used herein, the “backbone amino acid sequence” of the IFN refers toany portion of the IFN protein or fragment excluding the portions of theIFN that binds to the IFNAR1 or IFNAR2 subunits of the Type 1 interferonreceptor. In some embodiments, the backbone sequence of the IFNcomprises one or more substitution mutations. In some embodiments, oneor more substitution mutations are made in the backbone sequence of theIFN such that residue 23 is A, residue 26 is P, residue 44 is D, residue52 is Q, residue 53 is A, residue 55 is S, residue 83 is E, residue 101is T, residue 104 is V, residue 105 is G, residue 107 is E, or residue125 is E.

In some embodiments, the one or more substitution mutations are selectedto enhance one or more properties (e.g., folding, expression, and/orintracellular stability) of the polypeptide or of the fusion proteinand/or the protein domain. In some embodiments the substitutionmutations are selected to enhance the stability of the trimeric complexformed between the IFN (or protein domain of IFN) and the IFNAR1 andIFNAR2 subunits. Increasing the stability of said trimeric complex mayresult in a trimeric complex with features such as increased resistanceto internalization, decreased trafficking to the lysosome, and/orresistance to degradation. Any of the foregoing features may increasethe signaling (e.g., strength or duration) through the IFNAR signalingpathway and enhance the anti-proliferative effects of the chimericactivator.

As used herein, a “mutation” refers to a change in a nucleotide sequencerelative to a wild-type form of a gene. A change in the nucleotidesequence may or may not lead to a change in the amino acid sequence, thethree-dimensional structure of the protein, and/or the activity of theprotein, relative to the wild-type form of the protein. In someembodiments a mutation may be a naturally occurring variant of the gene.In some embodiments a mutation results in a single amino acidsubstitution, two or more amino acid substitutions, one or moredeletions, one or more insertions, or any combination of two or morethereof, in the protein sequence of any portion of the fusion proteinsor chimeric activators described herein.

In some embodiments, the amino acid sequence of the polypeptide of thechimeric activator is at least 80% (e.g., 85%, 90%, 95%, 98%, 99%)identical to IFN-α2. In some embodiments, the amino acid sequence of achimeric activator is at least 80% (e.g., 85%, 90%, 95%, 98%, 99%)identical to the sequence of one or more chimeric activators describedherein. The “percent identity” of two amino acid sequences is determinedusing the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad.Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into theNBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol.Biol. 215:403-10, 1990. BLAST protein searches can be performed with theXBLAST program, score=50, wordlength=3 to obtain amino acid sequenceshomologous to the protein molecules of interest. Where gaps existbetween two sequences, Gapped BLAST can be utilized as described inAltschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used.

In some embodiments, aspects of the present disclosure are based on therecognition that modulating the binding affinity of a protein domain ofthe fusion protein to each of the receptor subunits of a multimeric(e.g., heterodimeric) receptor (e.g., the IFNAR1 and IFNAR2 subunits ofIFNAR) results in enhanced signaling through the receptor (e.g., Type 1interferon receptor). Such modulation can also result in enhancedspecificity of the desired activity of the chimeric activator to thetarget cell and reduced activity on side effect cells. Aspects of thedisclosure relate to protein domains that bind a multimeric targetreceptor (e.g., IFNAR) and comprise one or more mutations to modulatethe binding affinity of the polypeptide to the receptor subunits (e.g.,IFNAR1 and IFNAR2 subunits of IFNAR).

Binding or interaction between the protein domain of the fusion proteinand the subunits of the multisubunit signaling receptor may induce asignaling transduction pathway in the cell that results in a desiredactivity in the cell (e.g., inhibit or prevent proliferation, inducecytotoxicity, induce or repress gene expression). In some embodiments,binding of the polypeptide portion of the chimeric activator to IFNARresults in a desired effect on the cell expressing IFNAR. In someembodiments, the desired effect is an anti-proliferative effect. Theanti-proliferative effect can be measured directly by quantifyingcellular proliferation by flow cytometry, cell counting, monitoringcellular metabolism or gene expression, or other methods known in theart. Binding of the polypeptide to IFNAR can be assessed by any methodknown in the art, including, without limitation, internal reflectionfluorescence microscopy, reflectance interference detection, assessingIFNAR subunit conformational change, assessing Jak/Stat signalingpathway activation, assaying production of one or more cytokine inducedby IFN or expression of any one or more genes regulated by IFN.

Aspects of the disclosure relate to mutations that effect the bindingaffinity of a polypeptide or protein domain to a receptor or antigen. Asused herein, “binding affinity” refers to the apparent associationconstant or K_(A). The K_(A) is the reciprocal of the dissociationconstant (K_(D)). The protein domain of a fusion protein describedherein may have a binding affinity (K_(D)) of at least 10⁻⁵, 10⁻⁶, 10⁻⁷,10⁻⁸, 10⁻⁹, 10⁻¹⁰ M, or lower to one or more subunits of the multimericsignaling receptor. An increased binding affinity corresponds to adecreased K_(D). Higher affinity binding of protein domain for a firstmolecule relative to a second molecule can be indicated by a higherK_(A) (or a smaller numerical value K_(D)) for binding the firstmolecule than the K_(A) (or numerical value K_(D)) for binding thesecond molecule. In some embodiments, mutations in the protein domainmay result in altering (increasing or decreasing) the binding affinityof the protein domain to one or more subunits of the multimericsignaling receptor. In some embodiments, the binding affinity of theprotein domain to one subunit of the multimeric receptor issubstantially the same as the binding affinity of the protein domain toanother subunit of the multimeric receptor.

In some embodiments, without the one or more amino acid substitutions, aprotein domain binds to a first subunit (e.g., the IFNAR2 subunit) witha higher binding affinity relative to the binding affinity of theprotein domain to the a second subunit (e.g., the IFNAR1 subunit).Mutations, such as substitutions, can be made to increase or decreasethe binding affinity of the polypeptide to the first subunit (e.g., theIFNAR2 subunit). In some embodiments, the binding affinity of theprotein domain to the first subunit (e.g., the IFNAR2 subunit) isreduced by at least 1.5-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9, 10-, 20-, 50-,100-fold or more relative to the protein domain that does not containthe mutation(s). In some embodiments, the binding affinity of thepolypeptide to the first subunit (e.g., the IFNAR2 subunit) is increasedby at least 1.5-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9, 10-, 20-, 50-, 100-foldor more relative to the protein domain that does not contain themutation(s). Mutations can be made to increase or decrease the bindingaffinity of the protein domain to the second subunit (e.g., the IFNAR1subunit). In some embodiments, the binding affinity of the proteindomain to the second subunit (e.g., the IFNAR1 subunit) is increased byat least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9, 10-, 20-, 50-, 100-fold or morerelative to the protein domain that does not contain the mutation(s). Insome embodiments, the binding affinity of the polypeptide to the secondsubunit (e.g., the IFNAR1 subunit) is reduced by at least 2-, 3-, 4-,5-, 6-, 7-, 8-, 9, 10-, 20-, 50-, 100-fold or more relative to theprotein domain that does not contain the mutation(s). In someembodiments, mutations are made to decrease the binding affinity of thepolypeptide to the first subunit (e.g., the IFNAR2 subunit) and toincrease the binding affinity of the polypeptide to the second subunit(e.g., the IFNAR1 subunit). In some embodiments, the binding affinity ofthe mutant polypeptide to the first subunit (e.g., the IFNAR2 subunit)is substantially equal to the binding affinity of the mutant polypeptideto the second subunit (e.g., the IFNAR1 subunit). In some embodiments,the binding affinity of the mutant polypeptide to the first subunit(e.g., the IFNAR2 subunit) is within a range of between 10:1 and 1:10(e.g., between 5:1 and 1:5, between 2:1 and 1:2, or within a range of+/−50%) of the binding affinity of the mutant polypeptide to the secondsubunit (e.g., the IFNAR1 subunit). FIG. 3 illustrates examples offusion proteins in which two mutations are introduced into a proteindomain (INFα), with one mutation increasing binding affinity for onesubunit, such as IFNAR1, and a second mutation decreasing bindingaffinity for the other receptor subunit, such as IFNAR2. The bindingaffinity of the resulting INFα for each receptor subunit isapproximately equal.

In some embodiments, one or more substitution mutations may be made inthe amino acid sequence of the protein domain that binds to IFNAR inorder to modulate the binding affinity to IFNAR1 or IFNAR2. Substitutionmutations can be selected from residues L30, R145, M149, E59, H58, andR150. In some embodiments, the one or more substitution mutations areselected from L30A, R145A, M149A, E59A, H58A, and R150A. In someembodiments, the IFN-α comprises mutations H58A and R150A. In someembodiments, the IFN-α comprises mutations E59A and M149A.

In some embodiments, the binding affinity of the protein domain of thefusion protein to the multimeric receptor is lower than the bindingaffinity of the antibody variable region element to the cell surfaceantigen. In some embodiments, the binding affinity of the protein domainof the fusion protein to IFNAR is lower than the binding affinity of theanti-EGFRvIII antibody element to EGFRvIII. In some embodiments, thebinding affinity of the protein domain to the multimeric receptor (e.g.,IFNAR) is at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9, 10-, 20-, 50-,100-fold or more lower than the binding affinity of the antibodyvariable region element to the cell surface antigen (e.g., anti-EGFRvIIIantibody element to EGFRvIII). In some embodiments, the binding affinityof the protein domain to IFNAR1 subunit, IFNAR2 subunit or both IFNAR1and IFNAR2 subunits may be modulated (increased or decreased) to reduceundesired activity on side effect cells. In some embodiments, modulatingthe binding affinity of the polypeptide results in enhanced specificityof the activity of a chimeric activator to a target cell (e.g., a cellexpressing EGFRvIII).

Binding affinity (or binding specificity) can be determined by a varietyof methods including equilibrium dialysis, equilibrium binding, gelfiltration, ELISA, surface plasmon resonance, or spectroscopy (e.g.,using a fluorescence assay).

Antibody Variable Region Elements that Bind Cell Surface Antigens

Aspects of the disclosure provide fusion proteins comprising an antibodyvariable region element that binds to a cell surface antigen. Theantibody variable region element may also be referred to as a targetingelement. As used herein, a “cell surface antigen” refers to a moleculethat is present on the surface of a cell and may identify the cell as atarget cell. Any protein or fragment thereof that is present on thesurface of the cell may be used as a cell surface antigen and bound byan antibody variable region element. In some embodiments, the cellsurface antigen is a protein that indicates the cell is a cancer cell(i.e., is a cancer cell marker). In some embodiments, the cell surfaceantigen is EGFRvIII. In other embodiments, the cell surface antigen maybe the tumor specific glycolipid GD2 or the B cell marker CD20, or anyother cell surface molecule characteristic of a tumor cell.

The term antibody variable region element encompasses any antibody orfragment thereof, such as a full length immunoglobulin molecule, or anantigen-binding fragment thereof (such as Fab, Fab′, F(ab′)2, Fv),single chain (scFv), mutant thereof, fusion protein comprising anantibody portion, humanized antibody, chimeric antibody, diabody, linearantibody, single chain antibody, multispecific antibody (e.g.,bispecific antibodies) and/or any other modified configuration of theimmunoglobulin molecule that comprises an antigen recognition site ofthe required specificity. An antibody is capable of binding to a cellsurface antigen, such as a cell surface receptor, through at least oneantigen recognition site located at the variable region of theimmunoglobulin molecule. In some embodiments, the antibody of the fusionproteins described herein is a scFv that binds to EGFRvIII (e.g.,MR1-1). In some embodiments, the antibody of the fusion proteindescribed herein is a full length immunoglobulin or fragment thereofthat binds to EGFRvIII. In some embodiments, the antibody binding toEGFRvIII targets the chimeric activator to the desired target cell. Insome embodiments, the target cell is a cancer cell.

Also within the scope of the present disclosure are antibodies that arederived from a parent antibody that is capable of binding to a cellsurface antigen (e.g., EGFRvIII). The parent antibodies may specificallybind a desired epitope of EGFRvIII, for example, an epitope that ispresent on EGFRvIII. In some embodiments, the epitope to which theantibody binds is present on EGFRvIII but absent on full length EGFR. Insome embodiments, the antibody is the MR1-1 scFv antibody. In someembodiments, the epitope comprises a novel glycine residue that isformed by the deletion of exons 2-7 in producing EGFRvIII.

One or more parent antibodies that may be used for constructing fusionproteins described herein can be naturally occurring antibodies (e.g.,an antibody derived from a human, mouse, rat, rabbit, horse, or sheep),genetically engineered antibodies (e.g., humanized antibodies, chimericantibodies), or antibodies derived from a natural or synthetic antibodylibrary. In some embodiments, the antibody is a full length monoclonalantibody. In some embodiments, the antibody is a single chain antibody(svFc). In some embodiments, the svFc is MR1-1.

In some examples, the antibody can be an affinity matured antibody,which refers to an antibody having one or more modifications in one ormore CDRs or framework regions (FRs) as compared to the unmodifiedparent antibody, leading to an improvement in the affinity of theantibody for the target antigen. Preferred affinity matured antibodiesmay have nanomolar or even picomolar affinities for the target antigen.Affinity maturation of an antibody can be performed by various methodsknown in the art, including by variable domain shuffling (see, e.g.,Marks et al. 1992, Bio/Technology 10:779-783), random mutagenesis of CDRand/or FR residues (see, e.g., Barbas et al., 1994, Proc Nat. Acad. Sci,USA 91:3809-3813; Schier et al., 1995, Gene 169:147-155; Yelton et al.,1995, J. Immunol. 155:1994-2004; Jackson et al., 1995, J. Immunol.154(7):3310-9; and Hawkins et al, 1992, J. Mol. Biol. 226:889-896). Theparent antibodies can be of any class, such as IgD, IgE, IgG, IgA, orIgM, or a sub-class thereof, or a single chain antibody, such as a scFv.

In some embodiments, the antibody variable region element of the fusionprotein specifically or selectively binds to the cell surface antigen.In some embodiments, the anti-EGFRvIII antibody of the fusion proteinspecifically or selectively binds to EGFRvIII. An antibody with“specific binding” reacts or associates more frequently, more rapidly,with greater duration and/or with greater affinity with the cell surfaceantigen than it does with a different molecule. The antibody variableregion element may “specifically bind” to the cell surface antigen withgreater affinity, avidity, more readily, and/or with greater durationthan it binds to other substances. “Specific binding” does notnecessarily require (although it can include) exclusive binding.Generally, but not necessarily, reference to binding means preferentialbinding. In some examples, an antibody that “specifically binds” to atarget antigen or an epitope thereof may not bind to other antigens orother epitopes in the same antigen. In some embodiments, the fusionproteins described herein specifically bind to EGFRvIII. In someembodiments, the fusion proteins do not bind to full length EGFR. Otherantibodies that specifically bind to EGFRvIII are compatible for use inthe fusion proteins described herein and are known in the art, forexample in Modjtahedi et al. 2003 Int. J. of Cancer 105(2) and PCTApplication No. WO 2001/062931.

In some embodiments, a fusion protein as described herein has a bindingaffinity for EGFRvIII or epitopes thereof such that the fusion proteinis targeted to a cell with EGFRvIII present on the cell surface. In someembodiments, the binding affinity of the fusion protein to EGFRvIII ishigher than the binding affinity of the fusion protein to full lengthEGFR. In some embodiments, the binding affinity of the fusion protein toEGFRvIII is at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91,100, 500, 1000, 10,000 or 10⁵ fold higher than the binding affinity ofthe chimeric activator to full length EGFR.

In one example, the anti-EGFRvIII antibody of the chimeric activatordescribed herein is a scFv that selectively binds to EGFRvIII. In someembodiments, the scFv that binds to EGFRvIII is MR1-1. In someembodiments, the antibody that binds to EGFRvIII is derived from MR1-1,which is described in PCT Application WO 2001/062931, incorporated byreference herein. The heavy chain and light chain sequences of MR1-1 areprovided below.

Heavy chain sequence of MR1-1 (SEQ ID NO: 15)EVQLVESGGGLVQPGGSLRLSCKVSGFTFSSYGMSWVRQAPGKGLEWVASISTGGYNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYSPYSYAMDYWGQGTTVTVS Light chain sequence of MR1-1 (SEQ ID NO: 16)DIQMTQSPSSLSASVGDRVTITCRASTDIDNDMNWYQQKPGQAPKLLIYEGNSLQSGVPSRFSSSGSGTDFTLTISSLQPEDFATYYCLQSWNV PLTFGQGTKLEIK

In other embodiments, the anti EGFRvIII antibody is the monoclonalantibody 806 or a fragment or variant therefor (see, for example, Johnset al. J. Biol. Chem. (2004) 279(29): 30375-84).

These may be linked by a suitable linker as illustrated herein. As iswell known in the art, within the antigen-binding portion of an antibodythere are complementarity determining regions (CDRs), which directlyinteract with the epitope of the antigen, and framework regions (FRs),which maintain the tertiary structure of the antigen-binding portion(see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain andthe light chain of IgG immunoglobulins, there are four framework regions(FR1-FR4) separated respectively by three complementarity determiningregions (CDR1-CDR3). The CDRs, and in particular the CDR3 regions, andmore particularly the heavy chain CDR3, are largely responsible forantibody specificity.

In some examples, the anti-EGFRvIII antibody is a functional variant ofMR1-1, which comprises up to 5 (e.g., 4, 3, 2, or 1) amino acid residuevariations in one or more of the CDR regions of MR1-1 and binds EGFRvIIIwith substantially similar affinity as MR1-1 (e.g., having a KD value inthe same order). In one example, the amino acid residue variations areconservative amino acid residue substitutions. As used herein, a“conservative amino acid substitution” refers to an amino acidsubstitution that does not alter the relative charge or sizecharacteristics of the protein in which the amino acid substitution ismade. Variants can be prepared according to methods for alteringpolypeptide sequence known to one of ordinary skill in the art such asare found in references which compile such methods, e.g., MolecularCloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. Conservative substitutions of aminoacids include substitutions made amongst amino acids within thefollowing groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G;(e) S, T; (f) Q, N; and (g) E, D.

In some embodiments, the anti-EGFRvIII comprises heavy chain CDRs thatare at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identicalto the corresponding heavy chain CDRs of MR1-1 and/or light chain CDRsthat are at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99%)identical to the corresponding light chain CDRs of MR1-1. In someembodiments, the anti-EGFRvIII antibody comprises a heavy chain variableregion that is at least 80% (e.g., 85%, 90%, 95%, or 98%) identical tothe heavy chain variable region of MR1-1 and/or light chain variableregion that is at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99%)identical to the light chain variable region of MR1-1.

In one example, the anti-EGFRvIII of the chimeric activator comprisesthe same heavy chain variable region (SEQ ID NO:15) and light chainvariable region (SEQ ID NO:16) as MR1-1.

In another example, the heavy chain variable region and light chainvariable region of MR1-1 can be combined with the constant regions of animmunoglobulin to produce a full length immunoglobulin or fragmentthereof that specifically binds to EGFRvIII. In some embodiments, theheavy chain CDR sequences and light chain CDR sequences of MR1-1 arecombined with the variable regions and constant regions of animmunoglobulin to produce a full length immunoglobulin or fragmentthereof that specifically binds to EGFRvIII. The constant regions can befrom any antibody isotype including IgG, IgA, IgM, IgE, IgD.

Also within the scope of the disclosure are antibody variable regionelements that bind any other cell surface antigens. In otherembodiments, the cell surface antigen may be the tumor specificglycolipid GD2 or the B cell marker CD20, or any other tumor ordisease-associated cell surface antigen. In some embodiments, the cellsurface antigen is GD2 and the antibody variable region element is the14.18 antibody or fragment or variant thereof (Ch14.18; see, for exampleBarker et al. Cancer Res. (1991) 51(1): 144-149). In some embodiments,the cell surface antigen is CD20 and the antibody variable regionelement is rituximab, or a fragment or variant thereof.

Linker

As used herein a “linker” refers to a polypeptide or a nucleic acid thatfunctions to attach two portions of a chimeric activator. The linker ofthe fusion proteins described herein connects the protein domain and theantibody variable region element. In some embodiments, the linkerconnects the C-terminal end of the antibody variable region element tothe N-terminal end of the protein domain. In other embodiments, thelinker connects the N-terminal end of the antibody variable regionelement to the C-terminal end of the protein domain.

A linker can comprise, for example 10 to 200 or more amino acids. Insome embodiments, the linker comprises 15-100, or 30-50 amino acids. Inother embodiments, the linker comprises, for example, 10 to 1000nucleotides or more. In some embodiments, the linker comprises 100-900,200-800, 300-700, 500-1000, or 700-1000 nucleotides. The amino acidsequence and/or length of the linker can be optimized for one or moredesired properties (e.g., separation of the polypeptide and the antibodyportions, prevention of self-binding of the portions of the chimericactivator). In some embodiments, the length of the linker allows forbinding of the protein domain to the multisubunit signaling receptor andbinding of the antibody variable region element to the cell surfaceantigen on the same cell. In some embodiments, the length of the linkerallows for binding of the protein domain to the multisubunit signalingreceptor and binding of the antibody variable region element to the cellsurface antigen at the same time.

In some embodiments, the linker has a net charge. In some embodiments,the linker has a negative net charge. In such embodiments, the aminoacid residues of the linker are selected from the group consisting ofglycine, serine, glutamate, and aspartate. In some embodiments, thelinker comprises repeats (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20more repeats) of GGGSE (SEQ ID NO:11), GSESG (SEQ ID NO:12), or GSEGS(SEQ ID NO:13). In some embodiments the linker comprises the sequence orSEQ ID NO:14: GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGG.

In some embodiments, the linker consists of a sequence with unchargedamino acids that promote flexibility and mitigate against formation of afolded structure, with glutamic acid or aspartic acid present atregularly spaced intervals. In some embodiments, the uncharged aminoacids are selected from glycine, serine, alanine, proline and threonine.In some embodiments, the uncharged amino acids are present in anon-repetitive pattern. Without wishing to be bound by any theory, theinterspersing uncharged amino acids in a non-repetitive pattern providesthat the nucleic acid encoding an expression construct for such a linkeris also non-repetitive and therefore may be less likely to undergointernal recombination events that lead to deletion. FIG. 2 illustratesexamples of linkers in which a charged amino acid is present with aregular spacing and the uncharged amino acids are present in anon-repeating pattern. In some embodiments, the linker comprisesnegatively charged amino acids with regular spacing interspersed withnon-charged amino acids, for example in non-regular spacing.

In other embodiments, the linker has a positive net charge. In suchembodiments, the amino acid residues of the linker are selected from thegroup consisting of lysine, histidine, and arginine.

Anti-EGFR/Anti-Type 1 IFN Receptor Fusion Proteins

In some embodiments, the fusion proteins described herein may comprise aprotein domain that binds to IFNAR, an antibody that binds to EGFRvIII,and a linker that connects the protein domain and the antibody. In someembodiments, the polypeptide comprises an interferon (IFN). In someembodiments, the antibody that binds EGFRvIII comprises a single chainantibody (scFv). In some embodiments, the scFv is MR1-1. In someembodiments, the C-terminus of the heavy chain variable region of MR1-1(SEQ ID NO:15) or the C-terminus of the light chain variable region ofMR1-1 (SEQ ID NO: 16) is fused to the linker. In some embodiments, thelinker is connected to N-terminus of the heavy chain variable region ofMR1-1 (SEQ ID NO:15) or the N-terminus of the light chain variableregion of MR1-1 (SEQ ID NO: 16). In some embodiments, the antibody thatbinds to EGFRvIII is the antibody 806. In some embodiments, theC-terminus of the antibody 806 is fused to the linker.

In some embodiments, the linker is connected to the N-terminus of theprotein domain. Alternatively, in other embodiments, the C-terminus ofthe protein domain is fused to the linker. Non-limiting examples oflinker sequences compatible for use in generating chimeric activatorsare described herein.

The heavy chain variable region of a svFc can be connected to the lightchain variable region of a svFc with a peptide linker, such as a peptidethat is rich in Gly and/or Ser residues, which are known in the art. Thepeptide linker can comprise, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more amino acid residues (e.g., up to 50 amino acid residues). Insome embodiments, the peptide linker can comprise 2-50, 5-25, or 5-20amino acids. In some embodiments, the peptide linker is a peptide richin glycine residues. In some embodiments, the peptide linker is rich inboth G and S.

Amino acid sequences for examples of fusion proteins are provided below.

MR1-1 IFNa 2-1b wt (SEQ ID NO: 1) consists of theheavy chain variable sequence of MR1-1, aglycine-serine linker (underlined), light chainvariable sequence of MR1-1, charged linker(underlined), and IFNα2-1b (bold) (SEQ ID NO: 1)EVQLVESGGGLVQPGGSLRLSCKVSGFTFSSYGMSWVRQAPGKGLEWVASISTGGYNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYSPYSYAMDYWGQGTTVTVSGGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASTDIDNDMNWYQQKPGQAPKLLIYEGNSLQSGVPSRFSSSGSGTDFTLTISSLQPEDFATYYCLQSWNVPLTFGQGTKLEIKGEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS CDLPQTHSLGSRRTLMLLPQMRRISPFSCLKDRHDFGFPQEEFDGNQFQKAQAISVLHEMIQQIFNLFSTKDSSAAWDETLLEKFYTELYQQLNDLEACVTQEVGVEETPLMNEDSILAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSLSTNLQERLRRKEHHHHHHMR1-1 IFNa 2-1b L30A (SEQ ID NO: 2) consists ofthe heavy chain variable sequence of MR1-1, aglycine-serine linker (underlined), light chainvariable sequence of MR1-1, charged linker(underlined), and IFNα2-b with L30A substitution (bold) (SEQ ID NO: 2)EVQLVESGGGLVQPGGSLRLSCKVSGFTFSSYGMSWVRQAPGKGLEWVASISTGGYNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYSPYSYAMDYWGQGTTVTVSGGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASTDIDNDMNWYQQKPGQAPKLLIYEGNSLQSGVPSRFSSSGSGTDFTLTISSLQPEDFATYYCLQSWNVPLTFGQGTKLEIKGEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS CDLPQTHSLGSRRTLMLIAQMRRISPFSCAKDRHDFGFPQEEFDGNQFQKAQAISVLHEMIQQIFNLFSTKDSSAAWDETLLEKFYTELYQQLNDLEACVTQEVGVEETPLMNEDSILAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSLSTNLQERLRRKEHHHHHH MR1-1 IFNa 2-1b R145A (SEQ ID NO: 3) consistsof the heavy chain variable sequence of MR1-1,a glycine-serine linker (underlined), lightchain variable sequence of MR1-1, chargedlinker (underlined), and IFNα2-b with R145A substitution (bold)(Seq ID NO: 3) EVQLVESGGGLVQPGGSLRLSCKVSGFTFSSYGMSWVRQAPGKGLEWVASISTGGYNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYSPYSYAMDYWGQGTTVTVSGGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASTDIDNDMNWYQQKPGQAPKLLIYEGNSLQSGVPSRFSSSGSGTDFTLTISSLQPEDFATYYCLQSWNVPLTFGQGTKLEIKGEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS CDLPQTHSLGSRRTLMLIAQMRRISPFSCLKDRHDFGFPQEEFDGNQFQKAQAISVLHEMIQQIFNLFSTKDSSAAWDETLLEKFYTELYQQLNDLEACVTQEVGVEETPLMNEDSILAVKKYFQRITLYLTEKKYSPCAWEVVAAEIMRSFSLSTNLQERLRRKEHHHHHH MR1-1 IFNa 2-1b E59A M149A (SEQ ID NO: 4)consists of the heavy chain variable sequenceof MR1-1, a glycine-serine linker (underlined),light chain variable sequence of MR1-1, chargedlinker (underlined), and IFNα2-b with E59A M149 substitutions (bold)(SEQ ID NO: 4) EVQLVESGGGLVQPGGSLRLSCKVSGFTFSSYGMSWVRQAPGKGLEWVASISTGGYNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYSPYSYAMDYWGQGTTVTVSGGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASTDIDNDMNWYQQKPGQAPKLLIYEGNSLQSGVPSRFSSSGSGTDFTLTISSLQPEDFATYYCLQSWNVPLTFGQGTKLEIKGEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS CDLPQTHSLGSRRTLMLIAQMRRISPFSCLKDRHDFGFPQEEFDGNQFQKAQAISVLHAMIQQIFNLFSTKDSSAAWDETLLEKFYTELYQQLNDLEACVTQEVGVEETPLMNEDSILAVKKYFQRITLYLTEKKYSPCAWEVVRAEIARSFSLSTNLQERLRRKEHHHHHH MR1-1 IFNa 2-1b H58A R150A (SEQ ID NO: 5)consists of the heavy chain variable sequenceof MR1-1, a glycine-serine linker (underlined),light chain variable sequence of MR1-1, chargedlinker (underlined), and IFNα2-b with H58A R150A substitutions (bold)(SEQ ID NO: 5) EVQLVESGGGLVQPGGSLRLSCKVSGFTFSSYGMSWVRQAPGKGLEWVASISTGGYNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYSPYSYAMDYWGQGTTVTVSGGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASTDIDNDMNWYQQKPGQAPKLLIYEGNSLQSGVPSRFSSSGSGTDFTLTISSLQPEDFATYYCLQSWNVPLTFGQGTKLEIKGEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS CDLPQTHSLGSRRTLMLLAQMRRISPFSCLKDRHDFGFPQEEFDGNQFQKAQAISVLAEMIQQIFNLFSTKDSSAAWDETLLEKFYTELYQQLNDLEACVTQEVGVEETPLMNEDSILAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMASFSLSTNLQERLRRKEHHHHHH

In the preceding sequences, the protein domain (IFN element) is based onthe following sequence, which is particularly useful as a domain withType 1 IFN activity:

SEQ ID NO: 18. CDLPQTHSLGSRRTLMLLAQMRRISPFSCLKDRHDFGFPQEEFDGNQFQKAQAISVLHEMIQQIFNLFSTKDSSAAWDETLLEKFYTELYQQLNDLEACVTQEVGVEETPLMNEDSILAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSLSTNLQERLRRKE

This polypeptide sequence may be fused to scFvs or other targetingmoieties such as whole antibodies, Fab fragments, minibodies, diabodies,camelid-based single-domain V regions, or non-antibody-based bindingunits such as short peptide aptamers or naturally occurring ligands.Targeting elements (antibody variable region elements) that bind totumor cells, tumor-associated cells such as stroma or endothelia, orvirus-infected cells may be useful as in fusion proteins with theprotein domain provided by SEQ ID NO: 18.

Preparation of Fusion Proteins

Methods known in the art, e.g., using standard recombinant technology,can be used for preparing the chimeric activators based on the presentdisclosure.

Antibody variable region elements can be obtained via routine technologyfrom suitable sources, e.g., PCR amplification from a suitable source.Similarly, protein domains may be obtained from a any suitable source.In one example, the DNA encoding an antibody variable region elementthat binds to a cell surface antigen and/or a protein domain that bindsto a multisubunit signaling receptor can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theantibody and/or protein domain.

Nucleotide sequences encoding one or more of portion of the fusionproteins as described herein can be cloned into one or more expressionvector, each nucleotide sequence being operably linked to a suitablepromoter. Alternatively, the nucleotides sequences can be operablylinked with a single promoter, such that each of the sequences isexpressed from the same promoter as a single protein. In someembodiments, the antibody variable region element is a full lengthimmunoglobulin, or fragment or derivative thereof, comprising twochains. In such examples, the two chains of the antibody portion of thefusion protein may be expressed separately (as two polypeptides) and canbe controlled by a common promoter. In other examples, the expression ofeach of the two chains of the fusion protein is under the control of adistinct promoter. In some embodiments, the nucleotide sequencesencoding the two chains of the fusion protein are cloned into twovectors, which can be introduced into the same or different cells. Whenthe two chains are expressed in different cells, each of them can beisolated from the host cells expressing such and the two isolated chainscan be mixed and incubated under suitable conditions allowing for theformation of the chimeric activator.

Generally, a nucleic acid sequence encoding one or all chains of achimeric activator can be cloned into a suitable expression vector inoperable linkage with a suitable promoter using methods known in theart. The selection of expression vectors/promoter would depend on thetype of host cells for use in producing the antibodies.

A variety of promoters can be used for expression of the fusion proteins(e.g., chimeric activators) described herein. In some embodiments, thefusion proteins are expressed in Pichia pastoris and the promoter is theU6 promoter or the AOX promoter (see, for example, Daly et al. J. Mol.Recognoit. (2005) 18(2) p 119-38. Other examples of promoters include,without limitation, including, but not limited to, cytomegalovirus (CMV)intermediate early promoter, a viral LTR such as the Rous sarcoma virusLTR, HIV-LTR, HTLV-1 LTR, the simian virus 40 (SV40) early promoter, E.coli lac UV5 promoter, and the herpes simplex tk virus promoter.

Regulatable promoters can also be used, such as regulatable promotersthat include a repressor with the operon can be used. In one embodiment,the lac repressor from E. coli can function as a transcriptionalmodulator to regulate transcription from lac operator-bearing mammaliancell promoters [M. Brown et al., Cell, 49:603-612 (1987)]; Gossen andBujard (1992); [M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551(1992)] combined the tetracycline repressor (tetR) with thetranscription activator (VP 16) to create a tetR-mammalian celltranscription activator fusion protein, tTa (tetR-VP 16), with thetetO-bearing minimal promoter derived from the human cytomegalovirus(hCMV) major immediate-early promoter to create a tetR-tet operatorsystem to control gene expression in mammalian cells. In one embodiment,a tetracycline inducible switch is used. The tetracycline repressor(tetR) alone, rather than the tetR-mammalian cell transcription factorfusion derivatives can function as potent trans-modulator to regulategene expression in mammalian cells when the tetracycline operator isproperly positioned downstream for the TATA element of the CMVIEpromoter (Yao et al., Human Gene Therapy). One particular advantage ofthis tetracycline inducible switch is that it does not require the useof a tetracycline repressor-mammalian cells transactivator or repressorfusion protein, which in some instances can be toxic to cells (Gossen etal., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc.Natl. Acad. Sci. USA, 92:6522-6526 (1995)), to achieve its regulatableeffects.

Additionally, the vector can contain, for example, some or all of thefollowing: a selectable marker gene, such as the neomycin gene forselection of stable or transient transfectants in mammalian cells;enhancer/promoter sequences from the immediate early gene of human CMVfor high levels of transcription; transcription termination and RNAprocessing signals from SV40 for mRNA stability; SV40 polyoma origins ofreplication and ColE1 for proper episomal replication; internal ribosomebinding sites (IRESes), versatile multiple cloning sites; and T7 and SP6RNA promoters for in vitro transcription of RNA. Suitable vectors andmethods for producing vectors containing transgenes are well known andavailable in the art.

Examples of polyadenylation signals useful to practice the methodsdescribed herein include, but are not limited to, human collagen Ipolyadenylation signal, human collagen II polyadenylation signal, andSV40 polyadenylation signal.

One or more vectors (e.g., expression vectors) comprising nucleic acidsencoding any of the fusion proteins may be introduced into suitable hostcells for producing the chimeric activator. The host cells can becultured under suitable conditions for expression of the fusion proteinor any polypeptide chain thereof. Such fusion proteins or polypeptidechains thereof can be recovered by the cultured cells (e.g., from thecells or the culture supernatant) via a conventional method, e.g.,affinity purification. If necessary, polypeptide chains of the fusionprotein can be incubated under suitable conditions for a suitable periodof time allowing for production of the chimeric activator.

Suitable host cells for use in preparing the fusion proteins describedherein can be any host cells known in the art that can be used forprotein production, including, but not limited to, bacterial cells,yeast cells, fungal cells, insect cells, plant cells, or mammaliancells.

The fusion proteins described herein can be produced in bacterial cells,e.g., E. coli cells. Alternatively, the chimeric activators can beproduced in eukaryotic cells. In one embodiment, the antibodies areexpressed in a yeast cell such as Pichia pastoris (see, e.g., Powers etal., 2001, J. Immunol. Methods. 251:123-35), Hanseula, or Saccharomyces.In another embodiment, the chimeric activators can be produced inmammalian cells. Mammalian host cells for expressing the moleculesinclude, but are not limited to, 293 cells (see, e.g., ATCC CRL-1573,American Type Culture Collection®, and Expi293F™ cells, LifeTechnologies™), Chinese Hamster Ovary (CHO cells) (including dhfr− CHOcells, described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA77:4216-4220, used with a DHFR selectable marker, e.g., as described inKaufman and Sharp, 1982, Mol. Biol. 159:601 621), lymphocytic celllines, e.g., NS0 myeloma cells and SP2 cells, COS cells, and a cell froma transgenic animal, e.g., a transgenic mammal. For example, the cell isa mammary epithelial cell.

In some embodiments, methods for preparing a chimeric activatordescribed herein involve a recombinant expression vector that encodesboth the heavy chain and the light chain of an anti-EGFRvIII antibody,wherein the heavy chain and the light chain can be fused (e.g., at theC-terminus) to a linker that is fused to a polypeptide that binds toIFNAR, as also described herein. In some embodiments, the methods forpreparing a chimeric activator described herein involve a recombinantexpression vector that encodes a single chain antibody (scFv) comprisingthe heavy chain variable region and the light chain variable region ofMR1-1, in which case the heavy chain variable region or the light chainvariable region is fused to a to a linker that is fused to a polypeptidethat binds to IFNAR, as also described herein. The recombinantexpression vector can be introduced into a suitable host cell (e.g., aP. pastoris cell) by a conventional method. Positive host cells thatexpress the fusion proteins can be selected and cultured under suitableconditions allowing for the expression of the one or more chains thatform the chimeric activator, which can be recovered from the cells orfrom the culture medium, as described in the Examples.

In other embodiments, the components of the fusion proteins areexpressed and/or produced independently and subsequently combined. Insome embodiments, the protein domain and the antibody variable regionelement are expressed each comprising a tag that allows covalentattachment of the components with a linker. Examples of such tagsinclude a SNAP tag or a CLIP tag (see, e.g., Keppler et al. Nat.Biotech. (2002) 21: 86-9).

Standard molecular biology techniques are used to prepare therecombinant expression vector, transfect the host cells, select fortransformants, culture the host cells and recovery of the fusionproteins from the culture medium. For example, the fusion proteins canbe isolated by affinity chromatography with a Protein A or Protein Gcoupled matrix.

Pharmaceutical Compositions

The fusion proteins (the encoding isolated nucleic acid, vectorscomprising such, or host cells comprising the vectors) as describedherein can be mixed with a pharmaceutically acceptable carrier(excipient) to form a pharmaceutical composition for use in treating atarget disease. A “pharmaceutically acceptable carrier” or“pharmaceutically acceptable excipient” refer to a carrier that must becompatible with the active ingredient of the composition (andpreferably, capable of stabilizing the active ingredient) and notdeleterious to the subject to be treated. Pharmaceutically acceptableexcipients (carriers) including buffers, which are well known in theart. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed.(2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.

The pharmaceutical compositions to be used in the present methods cancomprise pharmaceutically acceptable carriers, excipients, orstabilizers in the form of lyophilized formulations or aqueoussolutions. (Remington: The Science and Practice of Pharmacy 20th Ed.(2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover). Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations used, and may comprise buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrans; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

In some examples, the pharmaceutical composition described hereincomprises liposomes containing the fusion protein (or the encodingisolated nucleic acid) which can be prepared by methods known in theart, such as described in Epstein, et al., Proc. Natl. Acad. Sci. USA82:3688 (1985); Hwang, et al., Proc. Natl. Acad. Sci. USA 77:4030(1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes withenhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter.

In other examples, the pharmaceutical composition described herein canbe formulated in sustained-release format. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the fusion protein, which matrices arein the form of shaped articles, e.g., films, or microcapsules. Examplesof sustained-release matrices include polyesters, hydrogels (forexample, poly(2-hydroxyethyl-methacrylate), or poly(v nylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), sucrose acetate isobutyrate, andpoly-D-(+3-hydroxybutyric acid.

The pharmaceutical compositions to be used for in vivo administrationmust be sterile. This is readily accomplished by, for example,filtration through sterile filtration membranes. Therapeuticcompositions are generally placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

The pharmaceutical compositions described herein can be in unit dosageforms such as tablets, pills, capsules, powders, granules, solutions orsuspensions, or suppositories, for oral, parenteral or rectaladministration, or administration by inhalation or insufflation.

For preparing solid compositions such as tablets, the principal activeingredient can be mixed with a pharmaceutical carrier, e.g.,conventional tableting ingredients such as corn starch, lactose,sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalciumphosphate or gums, and other pharmaceutical diluents, e.g., water, toform a solid preformulation composition containing a homogeneous mixtureof a compound of the present disclosure, or a non-toxic pharmaceuticallyacceptable salt thereof. When referring to these preformulationcompositions as homogeneous, it is meant that the active ingredient isdispersed evenly throughout the composition so that the composition maybe readily subdivided into equally effective unit dosage forms such astablets, pills and capsules. This solid preformulation composition isthen subdivided into unit dosage forms of the type described abovecontaining from 0.1 to about 500 mg of the active ingredient of thepresent disclosure. The tablets or pills of the novel composition can becoated or otherwise compounded to provide a dosage form affording theadvantage of prolonged action. For example, the tablet or pill cancomprise an inner dosage and an outer dosage component, the latter beingin the form of an envelope over the former. The two components can beseparated by an enteric layer that serves to resist disintegration inthe stomach and permits the inner component to pass intact into theduodenum or to be delayed in release. A variety of materials can be usedfor such enteric layers or coatings, such materials including a numberof polymeric acids and mixtures of polymeric acids with such materialsas shellac, cetyl alcohol and cellulose acetate.

Suitable surface-active agents include, in particular, non-ionic agents,such as polyoxyethylenesorbitans (e.g., Tween™ 20, 40, 60, 80 or 85) andother sorbitans (e.g., Span™ 20, 40, 60, 80 or 85). Compositions with asurface-active agent will conveniently comprise between 0.05 and 5%surface-active agent, and can be between 0.1 and 2.5%. It will beappreciated that other ingredients may be added, for example mannitol orother pharmaceutically acceptable vehicles, if necessary.

Suitable emulsions may be prepared using commercially available fatemulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ andLipiphysan™. The active ingredient may be either dissolved in apre-mixed emulsion composition or alternatively it may be dissolved inan oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil,corn oil or almond oil) and an emulsion formed upon mixing with aphospholipid (e.g., egg phospholipids, soybean phospholipids or soybeanlecithin) and water. It will be appreciated that other ingredients maybe added, for example glycerol or glucose, to adjust the tonicity of theemulsion. Suitable emulsions will typically contain up to 20% oil, forexample, between 5 and 20%. The fat emulsion can comprise fat dropletsbetween 0.1 and 1.0 .im, particularly 0.1 and 0.5 .im, and have a pH inthe range of 5.5 to 8.0.

The emulsion compositions can be those prepared by mixing a chimericactivator with Intralipid™ or the components thereof (soybean oil, eggphospholipids, glycerol and water).

Pharmaceutical compositions for inhalation or insufflation includesolutions and suspensions in pharmaceutically acceptable, aqueous ororganic solvents, or mixtures thereof, and powders. The liquid or solidcompositions may contain suitable pharmaceutically acceptable excipientsas set out above. In some embodiments, the compositions are administeredby the oral or nasal respiratory route for local or systemic effect.

Compositions in preferably sterile pharmaceutically acceptable solventsmay be nebulised by use of gases. Nebulised solutions may be breatheddirectly from the nebulising device or the nebulising device may beattached to a face mask, tent or intermittent positive pressurebreathing machine. Solution, suspension or powder compositions may beadministered, preferably orally or nasally, from devices which deliverthe formulation in an appropriate manner.

Methods of Treatment

In some embodiments, the fusion proteins, the encoding isolated nucleicacid, vectors comprising such, or host cells comprising the vectors,described herein are useful for treating cancer associated withexpression of a target molecule (e.g., EGFRvIII, e.g., characterized byconstitutive EGFR signaling, unregulated cellular growth andproliferation, survival, invasive capacity, and/or angiogenesis).

To practice the method disclosed herein, an effective amount of apharmaceutical composition described herein can be administered to anindividual (e.g., a human) in need of the treatment via a suitableroute, such as intravenous administration, e.g., by intramuscular,intraperitoneal, intracerebrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, inhalation or topical routes. Theindividual to be treated by the methods described herein can be amammal, for example a human. Mammals include, but are not limited to,farm animals, sport animals, pets, primates, horses, dogs, cats, miceand rats. A human who needs the treatment may be a human patient having,at risk for, or suspected of having a cancer, such as a glioblastoma, asquamous cell carcinoma, a solid tumor, or other cancer or conditionassociated with a target molecule of interest. A subject having a cancercan be identified by routine medical examination, e.g., laboratorytests, organ functional tests, CT scans, or ultrasounds. A subjectsuspected of having a cancer might show one or more symptoms of thedisease/disorder. A subject at risk for the disease/disorder can be asubject having one or more of the risk factors for that cancer.

In some embodiments, the method for treating the individual with cancercomprises detecting whether the cancer expresses EGFRvIII. If expressionof EGFRvIII is detected, the individual may be administered an effectiveamount of the fusion protein described herein. Detection of whether acancer expresses EGFRvIII may be performed by any method known in theart or described herein. For example, a sample can be obtained from theindividual and the expression of EGFRvIII can be detected using methodsincluding, without limitation immunofluorescence, Western blotting,fluorescence in situ hybridization using a probe the binds to theEGFRvIII RNA, PCR amplification of the EGFR gene using oligonucleotidethat flank exons 2 and 7, sequencing of the EGFR genomic locus, orevaluating activity of EGFRvIII (e.g., constitutive signaling).

Cancer is a disease characterized by uncontrolled or aberrantlycontrolled cell proliferation and other malignant cellular properties(for example resulting from constitutive EGFR signaling). As usedherein, the term cancer includes, but is not limited to, the followingtypes of cancer: breast cancer; biliary tract cancer; bladder cancer;brain cancer including glioblastomas and medulloblastomas; cervicalcancer; choriocarcinoma; colon cancer; endometrial cancer; esophagealcancer; gastric cancer; hematological neoplasms including acutelymphocytic and myelogenous leukemia; T-cell acute lymphoblasticleukemia/lymphoma; hairy cell leukemia; chronic myelogenous leukemia,multiple myeloma; AIDS-associated leukemias and adult T-cellleukemia/lymphoma; intraepithelial neoplasms including Bowen's diseaseand Paget's disease; liver cancer; lung cancer; lymphomas includingHodgkin's disease and lymphocytic lymphomas; neuroblastomas; oral cancerincluding squamous cell carcinoma; ovarian cancer including thosearising from epithelial cells, stromal cells, germ cells and mesenchymalcells; pancreatic cancer; prostate cancer; rectal cancer; sarcomasincluding leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma,and osteosarcoma; skin cancer including melanoma, Merkel cell carcinoma,Kaposi's sarcoma, basal cell carcinoma, and squamous cell cancer;testicular cancer including germinal tumors such as seminoma,non-seminoma (teratomas, choriocarcinomas), stromal tumors, and germcell tumors; thyroid cancer including thyroid adenocarcinoma andmedullar carcinoma; and renal cancer including adenocarcinoma and Wilmstumor.

In certain embodiments, cancer is a colon carcinoma, a pancreaticcancer, a breast cancer, an ovarian cancer, a prostate cancer, asquamous cell carcinoma, a cervical cancer, a lung carcinoma, a smallcell lung carcinoma, a bladder carcinoma, a squamous cell carcinoma, abasal cell carcinoma, an adenocarcinoma, a sweat gland carcinoma, asebaceous gland carcinoma, a papillary carcinoma, a papillaryadenocarcinoma, a cystadenocarcinoma, a medullary carcinoma, abronchogenic carcinoma, a renal cell carcinoma, a hepatocellularcarcinoma, a bile duct carcinoma, a choriocarcinoma, a seminoma, aembryonal carcinoma, a Wilms' tumor, or a testicular tumor. In someembodiments, cancer is a glioblastoma. In some embodiments, cancer is asquamous cell carcinoma. Other cancers, for example carcinomas, will beknown to one of ordinary skill in the art.

“An effective amount” as used herein refers to the amount of the fusionprotein required to confer therapeutic effect on the subject, eitheralone or in combination with one or more other active agents. In someembodiments, the therapeutic effect is reduced cellular proliferation orcell viability of cells expressing EGFRvIII. Determination of whether anamount of the chimeric activator achieved the therapeutic effect wouldbe evident to one of skill in the art. Effective amounts vary, asrecognized by those skilled in the art, depending on the particularcondition being treated, the severity of the condition, the individualpatient parameters including age, physical condition, size, gender andweight, the duration of the treatment, the nature of concurrent therapy(if any), the specific route of administration and like factors withinthe knowledge and expertise of the health practitioner. These factorsare well known to those of ordinary skill in the art and can beaddressed with no more than routine experimentation. For administrationof any of the fusion proteins described herein, an individual may beadministered between 5 mcg-3 mg, 25 mcg-625 mcg, or approximately 125mcg of any of the fusion proteins described herein per week. Forrepeated administrations over several days or longer, depending on thecondition, the treatment is sustained until a desired suppression ofsymptoms occurs or until sufficient therapeutic levels are achieved toalleviate a target disease or disorder, or a symptom thereof. In someembodiments, dosing frequency is once a week, twice a week, three timesper week, four times per week, five times per week, every 2 weeks, every4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks,every 9 weeks, or every 10 weeks; or once every month, every 2 months,or every 3 months, or longer. The progress of this therapy is easilymonitored by conventional techniques and assays. The dosing regimen(including the antibody used) can vary over time.

Typically a clinician will administer a fusion protein, until a dosageis reached that achieves the desired result. In some embodiments, thedesired result is a reduction of cellular proliferation, cell viability,invasive capability, angiogenesis, or tumorigencity of a cell thatexpresses EGFRvIII. Methods of determining whether a dosage resulted inthe desired result would be evident to one of skill in the art.Administration of one or more chimeric activator can be continuous orintermittent, depending, for example, upon the recipient's physiologicalcondition, whether the purpose of the administration is therapeutic orprophylactic, and other factors known to skilled practitioners. Theadministration of a chimeric activator may be essentially continuousover a preselected period of time or may be in a series of spaced dose,e.g., either before, during, or after developing a target disease ordisorder.

As used herein, the term “treating” refers to the administration of acomposition including a fusion protein as described herein to anindividual, who has a cancer, a symptom of cancer, or a risk ofdeveloping cancer, with the purpose to cure, heal, alleviate, relieve,alter, remedy, ameliorate, improve, or affect the disease, a symptom, orthe risk of developing cancer.

Alleviating a disease/disorder, such as cancer, includes delaying thedevelopment or progression of the disease, or reducing disease severity.Alleviating the disease does not necessarily require curative results.As used therein, “delaying” the development of a target disease ordisorder means to defer, hinder, slow, retard, stabilize, and/orpostpone progression of the disease. This delay can be of varyinglengths of time, depending on the history of the disease and/orindividuals being treated. A method that “delays” or alleviates thedevelopment of a disease, or delays the onset of the disease, is amethod that reduces probability of developing one or more symptoms ofthe disease in a given time frame and/or reduces extent of the symptomsin a given time frame, when compared to not using the method. Suchcomparisons are typically based on clinical studies, using a number ofsubjects sufficient to give a statistically significant result.

“Development” or “progression” of a disease means initial manifestationsand/or ensuing progression of the disease. Development of the diseasecan be detectable and assessed using standard clinical techniques aswell known in the art. However, development also refers to progressionthat may be undetectable. For purpose of this disclosure, development orprogression refers to the biological course of the symptoms.“Development” includes occurrence, recurrence, and onset. As used herein“onset” or “occurrence” of a target disease or disorder includes initialonset and/or recurrence.

In some embodiments, the fusion protein described herein is administeredto a subject in need of the treatment at an amount sufficient to inhibitthe activity of one or both of the target antigens by at least 20%(e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) in vivo. In otherembodiments, the fusion protein is administered in an amount effectivein reducing the level of one or both target antigens by at least 20%(e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater).

The fusion proteins or nucleic acids encoding the fusion proteins may bedelivered using gene delivery vehicles. The gene delivery vehicle can beof viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy(1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, HumanGene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148).Expression of such coding sequences can be induced using endogenousmammalian or heterologous promoters and/or enhancers. Expression of thecoding sequence can be either constitutive or regulated.

Viral-based vectors for delivery of a desired polynucleotide andexpression in a desired cell are well known in the art. Exemplaryviral-based vehicles include, but are not limited to, recombinantretroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622;WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S.Pat. Nos. 5,219,740 and 4,777,127; GB Patent No. 2,200,651; and EPPatent No. 0 345 242), alphavirus-based vectors (e.g., Sindbis virusvectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross Rivervirus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitisvirus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), andadeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655). Administration of DNA linked to killed adenovirus asdescribed in Curiel, Hum. Gene Ther. (1992) 3:147 can also be employed.

Non-viral delivery vehicles and methods can also be employed, including,but not limited to, polycationic condensed DNA linked or unlinked tokilled adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992)3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989)264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S.Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO95/30763; and WO 97/42338) and nucleic charge neutralization or fusionwith cell membranes. Naked DNA can also be employed. Exemplary naked DNAintroduction methods are described in PCT Publication No. WO 90/11092and U.S. Pat. No. 5,580,859. Liposomes that can act as gene deliveryvehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos.WO 95/13796; WO 94/23697; WO 91/14445; and EP Patent No. 0524968.Additional approaches are described in Philip, Mol. Cell. Biol. (1994)14:2411, and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

The particular dosage regimen, i.e., dose, timing and repetition, usedin the method described herein will depend on the particular subject andthat subject's medical history.

In some embodiments, more than one chimeric activator, or a combinationof a chimeric activator and another suitable therapeutic agent, may beadministered to a subject in need of the treatment. The chimericactivator can also be used in conjunction with other agents that serveto enhance and/or complement the effectiveness of the agents.

Treatment efficacy for a target disease/disorder can be assessed bymethods well-known in the art.

Without further elaboration, it is believed that one of skill in the artcan, based on the above description, utilize the present disclosure toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever. All publicationscited herein are incorporated by reference for the purposes or subjectmatter referenced herein.

EXAMPLES Example 1 Design of Genetic Elements Encoding Fusion Proteinsand Insertion into Expression Vectors

The following sequences were generated and inserted into the expressionvector pPICZalpha A (Invitrogen/Life Technologies) by a combination ofcommercial DNA synthesis (Genscript, Inc.) and standard DNA cloning andassembly techniques. Specifically, the sequences below were placeddirectly after the sequence . . . AGA GAG GCT GAA GCT (SEQ ID NO: 19),which encodes the amino acids Arg Glu Ala Glu Ala (SEQ ID NO: 20) thatare part of the signal sequence that is removed by processing. At the 3′end of the SEQ ID NOs: 2, 6, 8, 9, and 10, the terminal sequence . . .“CAT CAC CAT CAC CAT CAC TAA” (SEQ ID NO: 21) was joined to the sequence“GTTTGTAGCC” (SEQ ID NO: 22) in the vector. Constructions were performedusing “Gibson assembly” (Gibson, D. G. “Enzymatic assembly ofoverlapping DNA fragments” Methods in Enzymology 498:349-362).

The fusion protein MR1-1 IFNα2-1b served as a “wild-type” and was usedas a point of comparison for derivatives or variants that had mutationsdesigned to decrease or increase binding to either INFα receptor 1(IFNAR1) or IFNα receptor 2 (IFNAR2). The interferon moiety of MR1-1IFNα2-1b was based on IFNα2a, but was significantly altered to improveits properties. The resulting interferon is distinct in sequence fromINFα2a and from the other human interferons, as well as from interferonsfrom other organisms.

The interferon-α moiety of the MR1-1 INFα2-1b protein had no mutationsfor the purpose of reducing INFα activity, but was re-engineeredrelative to natural human interferons in the following ways. The matureINFα moiety consisted of human INFα2a, but with an insertion of Asp atapproximately position 41 in the sequence Glu-Glu-Phe-Gly-Asn-Gln (SEQID NO: 23) . . . to yield Glu-Glu-Phe-Asp-Gly-Asn-Gln (SEQ ID NO: 24).This resulted in a renumbering of the sequence relative to INFα2asequence. The sequence of the IFN moiety of MR1-1 INFα2-1b alsocontained mutations of Arg23Lys, Leu26Pro, Glu53Gln, Thr54Ala, Pro56Ser,Asp86Glu, Ile104Thr, Gly106Glu, Thr110Glu, Lys117Asn, Arg125Lys, andLys136Thr. This set of mutations had the effect of enhancingproductivity of the fusion protein in Pichia pastoris. Production ofMR1-1 INFα2-1b was at least 3-fold greater than production ofMR1-1-INFα2a from Pichia pastoris. In addition, a significant fractionof the MR1-1-INFα2a underwent a cleavage near the C-terminus of theprotein that was not observed with the MR1-1 INFα2-1b protein.

About five independent P. pastoris Zeocin-resistant transformantscontaining plasmids designed to express MR1-1 INFα2-1b or MR1-1 INFα2awere tested for their ability to express full-length proteins insmall-scale (5 ml) cultures induced with methanol (BMMY medium).

After the transformation and plating on Zeocin plates, colonies wereincubated for three days then picked from the plate and re-plated on araster plate. The same colonies were labeled and grown in YPD Zeocin 200mcg/mL (30° C.) to make glycerol stocks by adding 500 microliters of 50%glycerol to 1 mL Pichia culture and freezing the stocks at −80° C. Eachcolony was also used to inoculate a 24-well plate to start proteinproduction screening. Typically, 4-6 transformants per construct wereanalyzed.

Transformants were grown in 1 mL BMGY media overnight at 30° C. at 140rpm in a 24-well plate sealed with air-pore tape. This allowedsufficient oxygen uptake by the cells. After overnight growth, theplate(s) were centrifuged at 1700 g for 5 minutes. The supernatant wasaspirated and replaced by 1 mL BMM+methanol media; cells wereresuspended in media. This initiated protein expression, and plates wereplaced back into the incubator (30° C. with 140 rpm). After 12-16 hours,the plates were centrifuged again at 3000 g for 5-10 minutes.Supernatants were stored and analyzed by SDS-PAGE gel to assess proteinexpression.

Supernatants were loaded directly into SDS-PAGE gels, and generally, 10microliters supernatant were mixed with 20 microliters 2× Novex buffer(Invitrogen). Samples were heated to 95° C. for 6 minutes, then loadedonto SDS-PAGE gels with 4-20% acrylamide gradient and run for 55 minutesat a constant current. Gels were stained in Coomassie Fluor Orange (LifeTechnologies) for about 40 minutes and analyzed. The best-expressingcolony per construct was selected for further evaluation. However,demonstrable variation in expression levels between colonies was notgenerally observed, so the improvement in expression of MR1-1 IFNα2-1brelative to MR1-1 INFα2a was ascribed to changes in the INFα proteinsequence, as the expression plasmids were otherwise identical.

Expression plasmids for mutated derivatives/variants of MR1-1 INFα2-1bwere constructed by standard techniques to introduce the followingmutations in the interferon moiety: Leu30Ala, Arg145Ala, His58Ala plusArg150Ala, and Glu59Ala Met149Ala. The following coding sequences wereused to express the corresponding fusion proteins.

MR1-1 IFNα 2-1b wt (SEQ ID NO: 6)GAAGTTCAATTGGTTGAGTCTGGTGGAGGTTTGGTTCAACCAGGAGGTTCCTTGAGATTGTCATGTAAAGTTTCTGGTTTTACTTTCTCTTCCTATGGAATGTCTTGGGTTAGACAAGCTCCTGGAAAGGGTTTGGAATGGGTTGCTTCCATCTCAACCGGTGGTTACAACACATATTACGCTGATTCCGTTAAAGGTAGATTCACTATCTCCAGAGATAACTCTAAGAACACTTTGTATTTGCAAATGAACTCTTTGAGAGCTGAAGATACTGCTGTTTACTATTGTGCGCGCGGTTACTCTCCATATTCTTACGCTATGGATTATTGGGGTCAAGGTACTACTGTTACTGTTTCTGGTGGAGGTGGAGGTTCAGGAGGTGGAGGAAGTGGAGGAGGTGGATCAGACATCCAGATGACACAATCACCATCTTCCTTGTCAGCTTCTGTTGGAGATAGAGTTACTATTACATGTAGAGCTTCCACTGACATCGATAACGACATGAATTGGTATCAGCAAAAACCTGGACAGGCGCCAAAGTTGTTGATCTACGAGGGTAACTCATTGCAATCTGGAGTTCCTTCCAGATTTTCATCTTCCGGTTCCGGAACAGATTTCACTTTGACAATCTCTTCCTTGCAGCCAGAAGACTTTGCTACTTATTACTGTTTGCAATCATGGAATGTTCCTTTGACATTCGGTCAAGGAACTAAATTGGAGATTAAGGGTGAAGGAGGGTCAGGTGAAGGTTCCTCCGGTGAGGGTTCCTCATCCGAAGGGGGAGGATCTGAAGGCGGTGGCTCTGAGGGTGGAGGTTCAGAGGGAGGGTCATGTGACTTGCCTCAAACTCATTCTTTGGGTTCTAGAAGAACTTTGATGTTGTTGGCTCAAATGAGAAGAATCTCTCCTTTCTCTTGTTTGAAGGACAGACATGACTTCGGTTTCCCTCAAGAGGAGTTCGACGGTAACCAATTCCAAAAGGCTCAAGCTATCTCTGTCTTGCATGAGATGATCCAACAAATCTTCAACTTGTTCTCTACTAAGGACTCTTCTGCTGCTTGGGACGAGACTTTGTTGGAGAAGTTCTACACTGAGTTGTACCAACAATTGAACGACTTGGAGGCTTGTGTCACTCAAGAGGTCGGTGTCGAGGAGACTCCTTTGATGAACGAGGACTCTATCTTGGCTGTCAAGAAGTACTTCCAAAGAATCACTTTGTACTTGACTGAGAAGAAGTACTCTCCTTGTGCTTGGGAGGTCGTCAGAGCTGAGATCATGAGATCTTTCTCTTTGTCTACTAACTTGCAAGAGAGATTGAGAAGAAAGGAGCATCACCATCACCATCACTAA MR1-1 IFNα 2-1b L30A(SEQ ID NO: 7)GAAGTTCAATTGGTTGAGTCTGGTGGAGGTTTGGTTCAACCAGGAGGTTCCTTGAGATTGTCATGTAAAGTTTCTGGTTTTACTTTCTCTTCCTATGGAATGTCTTGGGTTAGACAAGCTCCTGGAAAGGGTTTGGAATGGGTTGCTTCCATCTCAACCGGTGGTTACAACACATATTACGCTGATTCCGTTAAAGGTAGATTCACTATCTCCAGAGATAACTCTAAGAACACTTTGTATTTGCAAATGAACTCTTTGAGAGCTGAAGATACTGCTGTTTACTATTGTGCGCGCGGTTACTCTCCATATTCTTACGCTATGGATTATTGGGGTCAAGGTACTACTGTTACTGTTTCTGGTGGAGGTGGAGGTTCAGGAGGTGGAGGAAGTGGAGGAGGTGGATCAGACATCCAGATGACACAATCACCATCTTCCTTGTCAGCTTCTGTTGGAGATAGAGTTACTATTACATGTAGAGCTTCCACTGACATCGATAACGACATGAATTGGTATCAGCAAAAACCTGGACAGGCGCCAAAGTTGTTGATCTACGAGGGTAACTCATTGCAATCTGGAGTTCCTTCCAGATTTTCATCTTCCGGTTCCGGAACAGATTTCACTTTGACAATCTCTTCCTTGCAGCCAGAAGACTTTGCTACTTATTACTGTTTGCAATCATGGAATGTTCCTTTGACATTCGGTCAAGGAACTAAATTGGAGATTAAGGGTGAAGGAGGGTCAGGTGAAGGTTCCTCCGGTGAGGGTTCCTCATCCGAAGGGGGAGGATCTGAAGGCGGTGGCTCTGAGGGTGGAGGTTCAGAGGGAGGGTCATGTGACTTGCCTCAAACTCATTCTTTGGGTTCTAGAAGAACTTTGATGTTGTTGGCTCAAATGAGAAGAATCTCTCCTTTCTCTTGTGCTAAGGACAGACATGACTTCGGTTTCCCTCAAGAGGAGTTCGACGGTAACCAATTCCAAAAGGCTCAAGCTATCTCTGTCTTGCATGAGATGATCCAACAAATCTTCAACTTGTTCTCTACTAAGGACTCTTCTGCTGCTTGGGACGAGACTTTGTTGGAGAAGTTCTACACTGAGTTGTACCAACAATTGAACGACTTGGAGGCTTGTGTCACTCAAGAGGTCGGTGTCGAGGAGACTCCTTTGATGAACGAGGACTCTATCTTGGCTGTCAAGAAGTACTTCCAAAGAATCACTTTGTACTTGACTGAGAAGAAGTACTCTCCTTGTGCTTGGGAGGTCGTCAGAGCTGAGATCATGAGATCTTTCTCTTTGTCTACTAACTTGCAAGAGAGATTGAGAAGAAAGGAGCATCACCATCACCATCACTAA MR1-1 IFNα 2-1b R145A(SEQ ID NO: 8)GAAGTTCAATTGGTTGAGTCTGGTGGAGGTTTGGTTCAACCAGGAGGTTCCTTGAGATTGTCATGTAAAGTTTCTGGTTTTACTTTCTCTTCCTATGGAATGTCTTGGGTTAGACAAGCTCCTGGAAAGGGTTTGGAATGGGTTGCTTCCATCTCAACCGGTGGTTACAACACATATTACGCTGATTCCGTTAAAGGTAGATTCACTATCTCCAGAGATAACTCTAAGAACACTTTGTATTTGCAAATGAACTCTTTGAGAGCTGAAGATACTGCTGTTTACTATTGTGCGCGCGGTTACTCTCCATATTCTTACGCTATGGATTATTGGGGTCAAGGTACTACTGTTACTGTTTCTGGTGGAGGTGGAGGTTCAGGAGGTGGAGGAAGTGGAGGAGGTGGATCAGACATCCAGATGACACAATCACCATCTTCCTTGTCAGCTTCTGTTGGAGATAGAGTTACTATTACATGTAGAGCTTCCACTGACATCGATAACGACATGAATTGGTATCAGCAAAAACCTGGACAGGCGCCAAAGTTGTTGATCTACGAGGGTAACTCATTGCAATCTGGAGTTCCTTCCAGATTTTCATCTTCCGGTTCCGGAACAGATTTCACTTTGACAATCTCTTCCTTGCAGCCAGAAGACTTTGCTACTTATTACTGTTTGCAATCATGGAATGTTCCTTTGACATTCGGTCAAGGAACTAAATTGGAGATTAAGGGTGAAGGAGGGTCAGGTGAAGGTTCCTCCGGTGAGGGTTCCTCATCCGAAGGGGGAGGATCTGAAGGCGGTGGCTCTGAGGGTGGAGGTTCAGAGGGAGGGTCATGTGACTTGCCTCAAACTCATTCTTTGGGTTCTAGAAGAACTTTGATGTTGTTGGCTCAAATGAGAAGAATCTCTCCTTTCTCTTGTTTGAAGGACAGACATGACTTCGGTTTCCCTCAAGAGGAGTTCGACGGTAACCAATTCCAAAAGGCTCAAGCTATCTCTGTCTTGCATGAGATGATCCAACAAATCTTCAACTTGTTCTCTACTAAGGACTCTTCTGCTGCTTGGGACGAGACTTTGTTGGAGAAGTTCTACACTGAGTTGTACCAACAATTGAACGACTTGGAGGCTTGTGTCACTCAAGAGGTCGGTGTCGAGGAGACTCCTTTGATGAACGAGGACTCTATCTTGGCTGTCAAGAAGTACTTCCAAAGAATCACTTTGTACTTGACTGAGAAGAAGTACTCTCCTTGTGCTTGGGAGGTCGTCGCTGCTGAGATCATGAGATCTTTCTCTTTGTCTACTAACTTGCAAGAGAGATTGAGAAGAAAGGAGCATCACCATCACCATCACTAA MR1-1 IFNα2-1b E59A M149A (SEQ ID NO: 9)GAAGTTCAATTGGTTGAGTCTGGTGGAGGTTTGGTTCAACCAGGAGGTTCCTTGAGATTGTCATGTAAAGTTTCTGGTTTTACTTTCTCTTCCTATGGAATGTCTTGGGTTAGACAAGCTCCTGGAAAGGGTTTGGAATGGGTTGCTTCCATCTCAACCGGTGGTTACAACACATATTACGCTGATTCCGTTAAAGGTAGATTCACTATCTCCAGAGATAACTCTAAGAACACTTTGTATTTGCAAATGAACTCTTTGAGAGCTGAAGATACTGCTGTTTACTATTGTGCGCGCGGTTACTCTCCATATTCTTACGCTATGGATTATTGGGGTCAAGGTACTACTGTTACTGTTTCTGGTGGAGGTGGAGGTTCAGGAGGTGGAGGAAGTGGAGGAGGTGGATCAGACATCCAGATGACACAATCACCATCTTCCTTGTCAGCTTCTGTTGGAGATAGAGTTACTATTACATGTAGAGCTTCCACTGACATCGATAACGACATGAATTGGTATCAGCAAAAACCTGGACAGGCGCCAAAGTTGTTGATCTACGAGGGTAACTCATTGCAATCTGGAGTTCCTTCCAGATTTTCATCTTCCGGTTCCGGAACAGATTTCACTTTGACAATCTCTTCCTTGCAGCCAGAAGACTTTGCTACTTATTACTGTTTGCAATCATGGAATGTTCCTTTGACATTCGGTCAAGGAACTAAATTGGAGATTAAGGGTGAAGGAGGGTCAGGTGAAGGTTCCTCCGGTGAGGGTTCCTCATCCGAAGGGGGAGGATCTGAAGGCGGTGGCTCTGAGGGTGGAGGTTCAGAGGGAGGGTCATGTGACTTGCCTCAAACTCATTCTTTGGGTTCTAGAAGAACTTTGATGTTGTTGGCTCAAATGAGAAGAATCTCTCCTTTCTCTTGTTTGAAGGACAGACATGACTTCGGTTTCCCTCAAGAGGAGTTCGACGGTAACCAATTCCAAAAGGCTCAAGCTATCTCTGTCTTGCATGCGATGATCCAACAAATCTTCAACTTGTTCTCTACTAAGGACTCTTCTGCTGCTTGGGACGAGACTTTGTTGGAGAAGTTCTACACTGAGTTGTACCAACAATTGAACGACTTGGAGGCTTGTGTCACTCAAGAGGTCGGTGTCGAGGAGACTCCTTTGATGAACGAGGACTCTATCTTGGCTGTCAAGAAGTACTTCCAAAGAATCACTTTGTACTTGACTGAGAAGAAGTACTCTCCTTGTGCTTGGGAGGTCGTCAGAGCTGAGATCGCGAGATCTTTCTCTTTGTCTACTAACTTGCAAGAGAGATTGAGAAGAAAGGAGCATCACCATCACCATCACTAA MR1-1 IFNα2-1b H58A R150A (SEQ ID NO: 10)GAAGTTCAATTGGTTGAGTCTGGTGGAGGTTTGGTTCAACCAGGAGGTTCCTTGAGATTGTCATGTAAAGTTTCTGGTTTTACTTTCTCTTCCTATGGAATGTCTTGGGTTAGACAAGCTCCTGGAAAGGGTTTGGAATGGGTTGCTTCCATCTCAACCGGTGGTTACAACACATATTACGCTGATTCCGTTAAAGGTAGATTCACTATCTCCAGAGATAACTCTAAGAACACTTTGTATTTGCAAATGAACTCTTTGAGAGCTGAAGATACTGCTGTTTACTATTGTGCGCGCGGTTACTCTCCATATTCTTACGCTATGGATTATTGGGGTCAAGGTACTACTGTTACTGTTTCTGGTGGAGGTGGAGGTTCAGGAGGTGGAGGAAGTGGAGGAGGTGGATCAGACATCCAGATGACACAATCACCATCTTCCTTGTCAGCTTCTGTTGGAGATAGAGTTACTATTACATGTAGAGCTTCCACTGACATCGATAACGACATGAATTGGTATCAGCAAAAACCTGGACAGGCGCCAAAGTTGTTGATCTACGAGGGTAACTCATTGCAATCTGGAGTTCCTTCCAGATTTTCATCTTCCGGTTCCGGAACAGATTTCACTTTGACAATCTCTTCCTTGCAGCCAGAAGACTTTGCTACTTATTACTGTTTGCAATCATGGAATGTTCCTTTGACATTCGGTCAAGGAACTAAATTGGAGATTAAGGGTGAAGGAGGGTCAGGTGAAGGTTCCTCCGGTGAGGGTTCCTCATCCGAAGGGGGAGGATCTGAAGGCGGTGGCTCTGAGGGTGGAGGTTCAGAGGGAGGGTCATGTGACTTGCCTCAAACTCATTCTTTGGGTTCTAGAAGAACTTTGATGTTGTTGGCTCAAATGAGAAGAATCTCTCCTTTCTCTTGTTTGAAGGACAGACATGACTTCGGTTTCCCTCAAGAGGAGTTCGACGGTAACCAATTCCAAAAGGCTCAAGCTATCTCTGTCTTGGCTGAGATGATCCAACAAATCTTCAACTTGTTCTCTACTAAGGACTCTTCTGCTGCTTGGGACGAGACTTTGTTGGAGAAGTTCTACACTGAGTTGTACCAACAATTGAACGACTTGGAGGCTTGTGTCACTCAAGAGGTCGGTGTCGAGGAGACTCCTTTGATGAACGAGGACTCTATCTTGGCTGTCAAGAAGTACTTCCAAAGAATCACTTTGTACTTGACTGAGAAGAAGTACTCTCCTTGTGCTTGGGAGGTCGTCAGAGCTGAGATCATGGCATCTTTCTCTTTGTCTACTAACTTGCAAGAGAGATTGAGAAGAAAGGAGCATCACCATCACCATCACTAA

Example 2 Expression and Activity Testing of Unpurified Protein fromCulture Supernatants

The production and cell-based testing of the fusion proteins wereperformed as follows. Pichia pastoris strains were engineered withexpression vectors based on the plasmid pPICz-alpha, using the variouscoding sequences presented herein. Standard procedures were used in theplasmid construction and integration into Pichia. Proteins wereexpressed following in Pichia pastoris following the instructions in the“EasySelect™ Pichia Expression Kit” supplied by Invitrogen/LifeTechnologies. BMMY medium was used for expression, as it was found toreduce proteolysis of the desired products. Production was generally inthe range of about 10 micrograms of MR1-1-linker-INFα per ml of Pichiaculture supernatant. This fusion protein was the major protein inculture supernatant.

Expression of MR1-1-linker-human IFN-α2 was compared with theMR1-1-linker-human IFN-α2-1b protein to test for the effects of themutation of several amino acids relative to INFα2. It was found that thefusion protein containing INFα2-1b was produced with much lessproteolysis than a corresponding fusion protein with INFα2.

The various proteins were obtained from culture supernatant bycentrifugal removal of the cells, filtration of the supernatant througha 0.2 micron filter to remove remaining cells and sterilize thepreparation, concentration, and buffer exchange into Dulbecco's modifiedEagle's medium. Serial 10-fold dilutions of the protein in this form wasadded to wells containing either U87MG glioblastoma cells or U87MG cellsexpressing EGFRvIII, from about 10⁻⁶ to 10⁻¹²M. Cells were plated atabout 10,000 cells per well in a 96-well plate and incubated for 70hours at 37° C., 5% CO₂, in high-glucose DMEM with 10% FBS and PenStrep(100 IU penicillin and 100 ″ g/ml streptomycin). Cell number was thenmeasured using the WST-1 reagent (Roche).

During the 70-hour incubation, cell number appeared to increase about2.5-fold. For each series shown in FIGS. 5A-5F, readings were normalizedto the readings with no added fusion protein. Maximal readings weretherefore about 1, minimal readings were about 0.4, and thus an IC50could be calculated as the concentration of fusion protein giving anormalized reading of about 0.7. Based on this calculation approach, itcan be seen that the various fusion proteins show a significanttargeting effect, with much greater activity on U87MG-EGFRvIII cellsthan on U87MG cells. In particular, MR1-1-INFα2-1b(“wild-type”),MR1-1-INFα2-1b(Arg145Ala), and MR1-1-INFα2-1b(Glu59Ala Met149Ala) showedrespectively about 200-fold, 1000-fold, and at least 1,000-foldspecificity for the EGFRvIII-expressing cells. This level of cell-typespecificity is much greater than that observed in previous studies oftargeted, mutated INFα fusion proteins, as described in Cironi et al. (JBiol Chem [2008] 283(13):8469-76), and indicates that the furtherconceptual advances herein are useful in enhancing cell type-specificaction of fusion proteins.

Fusion proteins bearing the INFα L30A mutation or the INFαHis58Ala+Arg150Ala mutations did not show complete inhibition ofproliferation even at the highest concentration tested. Use of thesemutations is thus less preferred.

It is noteworthy that “wild-type” INFα2-1b showed a significanttargeting effect. This was not expected based on previous work. Withoutwishing to be bound by theory, it may be that the negatively chargedlinker attached to INFα2-1b effectively reduces the binding of INFα2-1bto its receptor. Comparison of the effect of INFα2-1b andMR1-1-linker-INFα2-1b on U87MG cells without EGFRvIII shows an IC50about 10⁻¹¹M and 10⁻¹⁰M respectively. It may be that the negativelycharged linker interacts with the positively charged receptor-bindingsurface of INFα2-1b, effectively reducing its on-rate in a manneranalogous to a mutation on this surface; according to this hypothesis,only a subset of molecules in which the linker is not interacting withINFα2-1b would be competent to bind. Thus, in some embodiments, theintroduction of negative charges into a linker in an <antibody Vregion>-<linker>-<positively charged signaling moiety> is beneficial.

Example 3 Scaled-Up Purification of a Novel MR1-1-INFalpha FusionProtein

The fusion protein MR1-1-INFα2-1b(R145A) was purified from engineeredPichia pastoris as follows. Many aspects of this procedure followed theprotocols given in manual for the EasySelect™ Pichia Expression Kit fromInvitrogen (Catalogue number K1740-01; Manual part number 25-0172),which defines formulas for the media used. The P. pastoris strain wasgrown to saturation in about 1 liter of BMGY medium, then centrifugedand resuspended in 2.4 liters of BMMY at a final OD of 0.5, distributedinto 10 two-liter Erlenmeyer flasks, and incubated for 48 hours at 30°C. with shaking at about 200 rpm and “Airport” air-permeable seals onthe top of the flasks. Cultures were then centrifuged at 10,000× gravityfor about 15 minutes, the supernatant harvested, filtered using (0.22um) filters, and transferred to a cold room. About 277 grams of(NH₄)₂SO₄ was added per liter and dissolved with agitation. Thissolution was incubated for 16 hours with gentle agitation, harvested bycentrifugation in 400 ml tubes at 20,000× gravity, then resuspended inHisTALON equilibration buffer (buffer kit catalog number 635651) inpreparation for purification based on cobalt affinity.

About 5 mls of TALON metal affinity resin was used for purification inaccordance with the manufacturer's instructions (Manual part numberPT1320-1). The protein was eluted in about 15 mls of HisTALON elutionbuffer, filter-sterilized, and further purified by FPLC. FIG. 2 shows atypical SDS-PAGE in which the purified protein is quantitated bycomparison with a bovine serum albumin standard. The final concentrationwas about 500 mcg/ml by this measurement.

Example 4 Activity of Purified Fusion Proteins on Diverse MammalianCells

The purified MR1-1-INFα2-1b(R145A) protein from Example 3 was tested forgrowth-inhibitory activity on U87MG glioblastoma cells or U87MG cellsexpressing EGFRvIII. Cell culture and assay procedures were essentiallyidentical to those described in Example 2. It was found that the ratioof IC50s for MR1-1-INFalpha2-1b(R145A)/INFalpha2 was about 18 on U87MGcells, but was about 0.122 on U87MG-EGFRvIII cells, indicating a nettargeting effect of about 148-fold.

A preparation of MR1-1-INFα2-1b that had been purified by His6-metalaffinity was tested for growth-inhibition of mouse Neuro 2a cells (N2acells). It was found that MR1-1-IFNα2-1b inhibited growth of the mousecells, although the IC50 for inhibition was about 50-fold greater thanfor murine INFα2. Human INFα2a had no detectable inhibitory activityagainst the mouse cells. These results are depicted in FIG. 8.

The activity against mouse cells is a valuable feature because humaninterferons generally do not activate mouse interferon receptors, whichprecludes biological testing of human interferons in natural mouseexperimental systems during the drug-development process.

Example 5 Toxicity and Pharmacokinetic Testing of Purified FusionProteins

The protein MR1-1-INFα2-1b(R145A) was purified as described in Example 3and tested for potential toxic effects in mice as follows. About 100micrograms of MR1-1-IFNα2-1b(R145A) protein in the physiologicallyacceptable carrier phosphate-buffered saline was injectedintraperitoneally into C57BL6 mice 5 days in a row (100 mcg/day). Threefemale mice were injected with the protein and two were injected withthe PBS vehicle; all were maintained as littermates. The mice wereobserved daily during the injection series and three days afterwards forsigns of toxicity including death or distress indicators such asdecreased activity, lethargy, decreased appetite, weight loss, poor bodycondition, abnormal or hunched body posture, poor grooming, respiratorydistress, eye squinting, nose bulge, ear position and/or whiskerchanges. All of the mice survived and no signs of toxicity were observedin the animals that received the MR1-1-IFNα2-1b(R145A) protein.

The protein MR1-1-INFα2-1b(no mutation) and MR1-1-INFα2-1b with amutation in the MR1-1 scFv (VH Ser52Arg, designed to block antigenbinding) were purified as described in Example 3 and tested for theirpharmacokinetic profile in mice as follows. About 12.5 micrograms ofeach fusion protein in phosphate-buffered saline was injectedintravenously into the tail vein of C57BL/6 mice (N=3 per dose group).Blood samples were withdrawn at 0, 1, 3, 7 and 24 hours, and theconcentration of MR1-1-INFα fusion proteins was determined by a standardELISA-type assay. Based on a comparison of the 7-hour and 24-hourtimepoints, the terminal serum half-lives of the MR1-1-INFα2-1b(nomutation) and MR1-1-INFα2-1b (mutant V region) fusion proteins wereabout 3 hours and 4 hours, respectively.

Example 6 Activity of Purified Fusion Proteins in an ImmunosuppressedTumor Model

To test the effect of the MR1-1-INFα2-1b(R145A) protein on tumors in amouse model, about 1 million oncogenic murine neural stem cells inapproximately 30% Matrigel (1:2.5 mixture Matrigel and PBS) wereinjected into the flanks of Nude C57Bl/6 mice and allowed to form smalltumors. The cells contained a knockout of the p16^(INK4a) gene andexpress EGFRvIII from a transgene (Bachoo et al. [2002] Cancer Cell1:269-277). Six mice per dose group were used. When the average tumorsize was about 100 mm³, the mice were divided into two groups with equalaverage tumor sizes. One group was injected intraperitoneally daily for5 days with about 100 micrograms of MR1-1-INFα2-1b(R145A) (135mcg/mouse/day; 150 microliters×0.9 mg/mL protein per mouse per day),while the other was injected intraperitoneally with a PBS vehiclecontrol.

The following results were observed. There was a statisticallysignificant difference in the growth rate of the tumors between the twogroups of mice, with the treated mice showing a cessation of tumorgrowth for about 1 week. Specifically, the tumor sizes in the mice thatreceived the fusion proteins were identical to the tumor sizes in micethat received the control on days 1 and 3. On day 5 the tumors in thetreated group of mice showed essentially no increase in size while thetumors in the untreated group of mice showed about a 50% increase. Onall subsequent days the tumors in the treated mice were, on average,smaller than the tumors in the untreated mice. Between days 7 and 9,tumor growth in the treated mice was observed to resume, possiblybecause the fusion protein had been cleared from the mouse and nolong-term immune response was possible because the mice are Nude micewith a defective immune system. FIG. 9.

To further test the effect of the MR1-1-INFα2-1b(R145A) protein ontumors in a mouse model, about 1 to 5 million U87MG-EGFRvIII cells inabout 30% Matrigel are injected into the flanks of Nude C57Bl/6 mice andallowed to form small tumors. Six mice per dose group are used. When theaverage tumor size is about 100 mm³, the mice are divided into groupswith equal average tumor sizes. One group was injected daily for 5 dayswith about 100 micrograms of MR1-1-INFα2-1b(R145A); another group wasinjected with MR1-1-INFα2-1b (no mutation); another group was injectedwith MR1-1-INFα2-1b(E59A M149A); and the final group was injected with aPBS vehicle control.

There was a statistically significant difference in the growth rate ofthe tumors between the control mice and the mice that had been treatedwith the fusion proteins, with the treated mice showing less dramaticgrowth and in some cases tumor shrinkage.

Example 7 Activity of Purified Fusion Proteins in a Syngeneic TumorModel

To test the effect of the fusion protein MR1-1-INFα2-1b (R145A) ontumors in an immunocompetent mouse model, about 1 million oncogenicmurine neural stem cells in 30% Matrigel were injected into the flanksof wild-type C57Bl/6 mice and were allowed to form small tumors. Thecells contained a knockout of the p16^(INK4a) gene and expressedEGFRvIII from a transgene (Bachoo et al. [2002] Cancer Cell 1:269-277).About six mice per dose group were used. When the average tumor size wasabout 100 mm³, the mice were divided into two groups with equal averagetumor sizes. One group was injected daily for 5 days with about 100micrograms of MR1-1-INFα2-1b (R145A). A second group was injected withMR1-1-INFα2-1b(no mutation). A third group was injected with a PBSvehicle control.

There was a statistically significant difference in the growth rate ofthe tumors between the treated groups of mice and the PBS-injectedcontrol mice, with the treated mice showing less dramatic growth and insome cases tumor shrinkage.

Example 8 Treatment of Human Patients with a Fusion Protein

A human patient with glioblastoma multiforme is treated with a fusionprotein as described herein, such as the MR1-1-INFα2-1b(R145A) fusionprotein.

The patient may be optionally tested to verify that his or her tumorcells express the EGFRvIII protein. Several diagnostic approaches arepossible, but two general approaches are based on the presence of thetumor nucleic acid or protein. The EGFRvIII protein is expressed as aresult of a deletion of exons 2 through 7 of the EGFR gene, resulting inexon 1 being spliced directly to exon 8. Because of this, the tumorcells express a novel genomic DNA segment that can be detected by PCR oftumor genomic DNA using primers that correspond to segments from exon 1and exon 8. DNA from normal tissue (that does not encode the EGFRvIIIvariant) will generally not be amplified because when all the exons arepresent, the distance between exon 1 and 8 is too large for efficientamplification under standard conditions. Similarly, mRNA from a tumorsample may be isolated and characterized by reverse transcriptase-PCR(RT-PCR) using primers corresponding to exon 1 and exon 8 or adownstream exon. In this case, the PCR products will typically include asignal from an intact EGFR mRNA that is present in normal cells that mayderive from tumor heterozygosity, normal adjacent tissue or normal cellsthat are part of the tumor such as stromal cells, fibroblasts, etc., butpresence of EGFRvIII mRNA will generate a distinct product that issmaller by 801 base pairs. The presence of such smaller products isdiagnostic of an EGFRvIII-type deletions/variants.

A patient's tumor may also be characterized by detection of the EGFRvIIIprotein, such as by antibody-based detection. For example, a tumorsample may be fixed and stained with an antibody that recognizes theEGFRvIII protein. For example, the variable regions or CDRs of the scFvMR1-1 V may be configured into a full length antibody with murine IgGconstant regions and used as a primary antibody. Probing with a EGFRvIIIspecific primary antibody can be followed by a secondary antibodydirected to the murine IgG constant regions and conjugated to ahistochemically detectable enzyme, such as horseradish peroxidase.Alternatively, protein may be extracted from a tumor or tumor cells andanalyzed by Western blot or ELISA assay using MR1-1 V regions to detectthe EGFRvIII protein.

In one treatment paradigm, a patient is first treated with surgery,followed by a combination of radiation therapy and temozolomide. Theradiation and temozolomide are administered according to standardpractice and the judgement of a physician, with a total dose of roughly60 Gy (Grays) or more delivered in doses of 1.8 to 2 Gy per day, 5 daysa week, and temozolomide delivered intravenously over 90 minutes indoses of 75 mg/m². The MR1-1-IFNα2-1b(R145A) fusion protein isadministered upon cessation of temozolomide treatment due to failure oftreatment or unacceptable side effects. Typical profiles for cessationof temozolomide would be a tumor volume of >0.5 cm³ but <1.5 cm³ and aplatelet count of <100,000/mm³, and a CTC grade 2 non-hematologictoxicity; or a tumor volume of <0.5 cm³ and a platelet count of<10,000/mm³, and a CTC grade 3 or 4 non-hematologic toxicity.

The fusion protein is administered intravenously in an appropriatesolution such as 5% dextrose over 1 to 4 hours. The dose is about 5 mcgto 3 mg or more per week, e.g., 25 mcg to 625 mcg per week; or about 125mcg/week, with administrations between 3 to 5 times per week. Thepatient is monitored for fever and other side effects. Without wishingto be bound by theory, the cessation of temozolomide results in enhancedsurvival of leukocytes that promote the activity of theMR1-1-INFα2-1b(R145A) fusion protein.

In a second treatment paradigm, a patient is first treated with surgery,followed by a combination of radiation therapy, temozolomide, andMR1-1-INFα2-1b(R145A) fusion protein. The radiation and temozolomide areadministered essentially as described above.

The fusion protein is administered intravenously in an appropriatesolution such as 5% dextrose over 1 to 4 hours. The dose is about 5 mcgto 3 mg or more per week, e.g., 25 mcg to 625 mcg per week or about 125mcg/week, with administrations between 3 to 5 times per week. Thepatient is monitored for fever and other side effects. Without wishingto be bound by theory, the combination of DNA damage in tumor cellsresulting from radiation plus temozolomide and the targeting of IFNαactivity on these cells is synergistic, results in enhanced killing ofthe tumor cells in the patient.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featurediscloses is only an example of a generic series of equivalent orsimilar features.

From the above description, one of skill in the art can easily ascertainthe essential characteristics of the present disclosure, and withoutdeparting from the spirit and scope thereof, can make various changesand modification of the disclosure to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

What is claimed is:
 1. A fusion protein comprising a Type 1 interferonprotein domain comprising the sequence of SEQ ID NO: 18 or a sequencewith one or two mutations relative to SEQ ID NO:18, the mutationsselected from the group consisting of: L30A, R145A, M149A, E59A, H58A,and R150A; an antibody variable region element comprising a heavy chainvariable region comprising the amino acid sequence provided by SEQ IDNO: 15 and a light chain variable region comprising the amino acidsequence provided by SEQ ID NO: 16, wherein the antibody variable regionelement binds to EGFRvIII; and a linker that connects the protein domainand the antibody variable region element.
 2. The fusion protein of claim1, wherein the fusion protein comprises one polypeptide chain.
 3. Thefusion protein of claim 1, wherein the Type 1 interferon protein domaincomprises mutations H58A and R150A.
 4. The fusion protein of claim 1,wherein the Type 1 interferon comprises mutations E59A and M149A.
 5. Thefusion protein of claim 1, wherein the linker connects the C-terminalend of the antibody variable region element to the N-terminal end of theprotein domain.
 6. The fusion protein of claim 1, wherein the linkerconnects the C-terminal end of the protein domain to the N-terminal endof the antibody or antibody variable region element.
 7. The fusionprotein of claim 1, wherein the linker is a peptide linker and has a netcharge.
 8. The fusion protein of claim 1, wherein the net charge of thelinker is negative.
 9. The fusion protein of claim 1, wherein the netcharge of the linker is positive.
 10. The fusion protein of claim 8,wherein the linker comprises amino acids selected from the groupconsisting of glycine, serine, glutamate, and aspartate.
 11. The fusionprotein of claim 9, wherein the linker comprises amino acids selectedfrom the group consisting of lysine, arginine, and histidine.
 12. Thefusion protein of claim 1, comprising the amino acid sequence of SEQ IDNO: 1 [MR1-1 IFNα2-lb WT].
 13. The fusion protein of claim 1, comprisingthe amino acid sequence of SEQ ID NO: 2 [MR1-1 IFNα2-1B L30A].
 14. Thefusion protein of claim 1, comprising the amino acid sequence of SEQ IDNO: 3 [MR1-1 IFNα2-1B R145A].
 15. The fusion protein of claim 1,comprising the amino acid sequence of SEQ ID NO: 4 [MR1-1 IFNα2-lb E59AM149A].
 16. An isolated fusion protein comprising the amino acidsequence of SEQ ID NO: 5 [MR1-1 IFNα2-lb H58A R150A].